SUMMARY A handful of tumor-derived cell lines form the mainstay of cancer therapeutic development, yielding drugs with impact typically measured as months to disease progression. To develop more effective breast cancer therapeutics and more readily understand their clinical impact, we constructed a functional metabolic portrait of 46 independently-derived breast cell lines. Our analysis of glutamine uptake and dependence identified a subset of triple negative samples that are glutamine auxotrophs. Ambient glutamine indirectly supports environmental cystine acquisition via the xCT antiporter, which is expressed on 1/3 of triple negative tumors in vivo. xCT inhibition with the clinically approved anti-inflammatory Sulfasalazine decreases tumor growth revealing a therapeutic target in breast tumors of poorest prognosis, and a lead compound for rapid, effective drug development.

SUMMARY1. Receptor currents in response to mechanical stimuli were recorded from hair cells in the excised epithelium of the bull-frog sacculus by the whole-cell, gigohm-seal voltage-clamp technique. The stimulus-dependent transduction current was separated from the cell's stimulus-independent K+ and Ca2+ currents; the K+ currents were blocked with an internal solution containing Cs+ while the Ca2+ current was reduced by holding the membrane potential below -70 mV. The temperature of the preparation was maintained at about 10 0C to slow the kinetics of the cells' transduction channels.2. Calibrated displacements of hair bundles of individual hair cells were made with a probe coupled by suction to the kinociliary bulb and moved with a piezoelectricbimorph stimulator. The root mean square noise of probe motion was less than 2 nm. The mean, I, and the variance, o.2, of the receptor current were measured from the response to saturating (± 0 5 ,m) displacements of the hair bundle. I was corrected for current offsets and o-2 for the transduction-independent background variance. 6. Control experiments show that transduction by the hair cell of two artifactual sources of hair-bundle stimulation, noisy or discontinuous motion of the probe, do not contribute substantially to the measured variance, o.2.7. Displacement-response curves are generally sigmoidal and symmetrical; they reasonably fit the predictions of a two-kinetic-state model, comprising one open state and one closed state. The estimated displacement-sensitive free energy, Z, is 5-7 + 1 1 kcal/mol ,m (mean+ S.D., n = 18).7-2 T. HOLTON AND A. J. HUDSPETH 8. Power-spectral densities of the receptor current were measured in response to steps of displacement. On the assumption of the two-kinetic-state model, the data suggest that a bundle displacement that gives 300 of maximum response corresponds to a mean lifetime of the channel in the open state of 0-9 ms at 9-5 0C or 130,us at 37 0C.

The response properties of hair cells and nerve fibers in the alligator lizard cochlea are frequency selective and tonotopically organized with longitudinal position in the organ. The lengths of the hair-cell hair bundles also vary monotonically with longitudinal position. In this study, quantitative measurements were made of the motion of individual hair bundles in an excised preparation of the cochlea stimulated at auditory frequencies. The angular displacement of hair bundles is frequency selective and tonotopically organized, demonstrating the existence of a micromechanical tuning mechanism.

TEXTAL is an automated system for building protein structures from electron-density maps. It uses pattern recognition to select regions in a database of previously determined structures that are similar to regions in a map of unknown structure. Rotation-invariant numerical values, called features, of the electron density are extracted from spherical regions in an unknown map and compared with features extracted around regions in maps generated from a database of known structures. Those regions in the database that match best provide the local coordinates of atoms and these are accumulated to form a model of the unknown structure. Similarity between the regions in the database and an uninterpreted region is determined firstly by evaluating the numerical difference in feature values and secondly by calculating the electron-density correlation coefficient for those regions with similar feature values. TEXTAL has been successful at building protein structures for a wide range of test electron-density maps and can automatically model entire protein structures in a few hours on a workstation. Models built by TEXTAL from test electron-density maps of known protein structures were accurate to within 0.6-0.7 A root-mean-square deviation, assuming prior knowledge of C(alpha) positions. The system represents a new approach to protein structure determination and has the potential to greatly reduce the time required to interpret electron-density maps in order to build accurate protein models.

The expression of heteroligomeric protein complexes for structural studies often requires a special coexpression strategy. The reason is that the solubility and proper folding of each subunit of the complex requires physical association with other subunits of the complex. The genomes of pathogenic mycobacteria encode many small protein complexes, implicated in bacterial fitness and pathogenicity, whose characterization may be further complicated by insolubility upon expression in Escherichia coli, the most common heterologous protein expression host. As protein fusions have been shown to dramatically affect the solubility of the proteins to which they are fused, we evaluated the ability of maltose binding protein fusions to produce mycobacterial Esx protein complexes. A single plasmid expression strategy using an N-terminal maltose binding protein fusion to the CFP-10 homolog proved effective in producing soluble Esx protein complexes, as determined by a small-scale expression and affinity purification screen, and coupled with intracellular proteolytic cleavage of the maltose binding protein moiety produced protein complexes of sufficient purity for structural studies. In comparison, the expression of complexes with hexahistidine affinity tags alone on the CFP-10 subunits failed to express in amounts sufficient for biochemical characterization. Using this strategy, six mycobacterial Esx complexes were expressed, purified to homogeneity, and subjected to crystallization screening and the crystal structures of the Mycobacterium abscessus EsxEF, M. smegmatis EsxGH, and M. tuberculosis EsxOP complexes were determined. Maltose binding protein fusions are thus an effective method for production of Esx complexes and this strategy may be applicable for production of other protein complexes.

SUMMARY1. Intracellular potentials were recorded with micropipettes from hair cells with free-standing stereocilia in the cochleae of anaesthetized alligator lizards.2. Wave forms of intracellular responses to click stimuli were classified into three types: hair cells, supporting cells, and untuned cells. We studied primarily the responses of hair cells to tonal stimuli.3. For most frequencies, f, and levels, P, of tone-burst stimuli, the response envelope of the receptor potential increases monotonically at the tone-burst onset, and decreases monotonically at tone-burst offset. Overshoot in the envelope of the response at the onset and offset of tone bursts is observed only for tone bursts of low f, high P, and short ( and V' components was measured by means of iso-voltage (iso-V0 and iso-V,)contours. Iso-V0 and iso-V' contours are V-shaped: the maximum sensitivity occurs at a characteristic frequency (c.f. ). The shapes of these contours near the c.f. depend on the values of V0 and V1 at which the contours were measured and are sharper for lower values of V1 and V1. The mean slopes of the low-and high-frequency sides of these contours are: -45-0 and + 85-1 dB/decade for iso-V0 contours (n = 26), and -33'6 and + 103'8 dB/decade for iso-V1 contours (n = 28).6. The receptor potential has non-linear properties. The magnitudes and phase angles of V0, 'l, V2, and V3 receptor-potential components were measured as a function of P for different f. The slopes of level functions (the dependence of log V0 and log V1I on log P) were measured at low levels for different f. T. HOLTON AND T. F. WEISSdiffering from c.f. by more than a half-octave, the slope for VT is between 1 and 2 with a mean of 1P3; the slope for V1 is about 1, i.e. V11 increases approximately linearly with P. For frequencies near c.f., the slopes for V0 and V1 are approximately 0-8 and 0 5, respectively, indicating the presence of a compressive non-linearity. An effect of this frequency-dependent non-linearity is to sharpen the frequency selectivity at lower response magnitudes for f near c.f.7. The receptor potential shows low-pass filtering. Whereas the maximum response magnitude of V0 (i.e. the saturation value measured at high P) does not depend on f, the maximum Vj decreases by approximately 20 dB/decade with increasing f. Values of IIJ1I measured at a constant value of V0 also decline by approximately 20 dB/decade.8. Many of the results are consistent with the predictions of a three-stage model of the generation of the receptor potential. The first stage, which represents the mechanical properties ofthe middle and inner ear, is a linear, time-invariant, band-pass filter; the second stage, which represents mechanoelectric transduction in hair cells, is a zero-memory non-linearity; the third stage, which represents the electrical properties of hair cells, is a linear, time-invariant, low-pass filter. However, the results differ from the model predictions for low-level acoustic stimuli near c.f., and indicate the presence of a frequency-dependent compr...

SUMMARY1. Receptor potentials of hair cells and spike discharges of cochlear nerve fibres were recorded with micropipettes from the free-standing region of the basilar papilla of anaesthetized alligator lizards in response to tones. In this region the hair-cell stereocilia are free-standing, i.e. they protrude directly into endolymph and are not in contact with a tectorial membrane.2. The frequency selectivity of hair-cell responses was measured by means of isovoltage contours of the d.c. ( VJ) and fundamental-a.c. (V1) component of the receptor potential, i.e. iso-Vo and iso-V1 contours. The frequency selectivity of the nerve-fibre discharge was measured by iso-rate (iso-A0) contours. Iso-V0, iso-V1 and iso-A0 contours are basically V-shaped with a characteristic frequency (c.f.) defined as the frequency at which minimum sound pressure (Pmin) is required to evoke the criterion value of the response.3. Receptor potential iso-V0 contours and neural iso-A0 contours have similar slopes: the mean slopes of the low-frequency sides (dB/decade) are -43 0 and -44-3; the slopes of the high-frequency sides are 85-0 and 802. The band widths of iso-V0 and iso-A0 contours away from c.f. are similar (mean values of Q30dB are 040 and 053, respectively). The band widths of iso-A0 contours near c.f. are narrower than those of iso-V0 contours (mean values of QlodB are 2-34 and 1-20, respectively). However, the shapes of the contours near c.f. depend on the iso-response criteria, and we have not determined whether or not iso-V0 and iso-A0 contours are similar near c.f. The shapes of iso-V1 contours differ from those of iso-V0 and iso-A0 contours.4. Nerve fibre c.f.s are tonotopically organized in the nerve, with lowest c.f.s recorded from fibres innervating the border of free-standing and tectorial regions, a region in which hair-cell stereocilia are longest, and the highest c.f.s recorded from fibres innervating the end of the free-standing region in which hair-cell stereocilia are shortest. The c.f. of nerve-fibre response (and by implication hair-cell response) is, therefore, correlated with the height of the stereociliary tuft.

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