Structure-activity relationships of boronic acid inhibitors of dipeptidyl peptidase IV. 1. Variation of the P2 position of Xaa-boroPro dipeptides.

A series of prolineboronic acid (boroPro) containing dipeptides were synthesized and assayed for their ability to inhibit the serine protease dipeptidyl peptidase IV (DPPIV). Inhibitory activity, which requires the (R)-stereoisomer of boroPro in the P1 position, appears to tolerate a variety of L-amino acids in the P2 position. Substitution at the P2 position which is not tolerated include the D-amino acids, alpha,alpha-disubstituted amino acids, and glycine. Specificity against DPPII and proline specific endopeptidase is reported. A correlation between the ability to inhibit DPPIV in cell culture and in the human mixed lymphocyte reaction is demonstrated. A synthesis of prolineboronic acid is reported as well as conditions for generating the fully unprotected boronic acid dipeptides in either their cyclic or acyclic forms.

Differentiation between renal cortex and medulla in the response to hypotension using localized 31P magnetic resonance spectroscopy.

Experiments were designed to test the hypothesis that renal medulla is more sensitive to hypoxia than is the cortex. Using the one-dimensional phase encoding technique to perform 31phosphorus magnetic resonance spectroscopy in a perfused porcine kidney preparation, cortex and medulla were differentiated on the basis of the unique resonance at 3 ppm found only in medulla. Hypotension-induced hypoxia reduced total renal oxygen consumption by 60%, and urine flow by 44%. Nonlocalized 31P MRS spectra showed that [ATP]/[Pi] ratio fell by 40%, and intrarenal pH by 0.1 unit. Virtually all of these changes could be accounted for by changes in the renal cortex, where initial [ATP]/[Pi] was higher than in medulla (1.16 vs. 0.68). In medulla [ATP]/[Pi] fell only 29% (n.s. versus control) and pH remained unchanged during hypotension. Thus the cortex appears to be more sensitive to hypoxia in this preparation, and observations fail to support the proposed hypothesis. They are consistent, however, with the greater capacity of medulla for anaerobic glycolysis. Localized 31P MRS provides improved noninvasive metabolic assessment of cold-preserved kidneys.

Short echo time magnetic resonance imaging of tendon.

RATIONALE AND OBJECTIVES: Current clinical magnetic resonance (MR) imaging studies provide almost no useful signal from normal tendon and have no clear advantage over other imaging modalities in the evaluation of tendon injuries. The authors believe that tendon MR signal may be T2-limited, and, if so, could be enhanced by short echo time (TE) pulse sequences. The relationship of tendon signal intensity and tendon infrastructure conspicuity to TE was assessed at three different field strengths. METHODS: Magnetic resonance imaging studies were performed on samples of normal equine tendon at 3 different field strengths with spin-echo or gradient-recalled-echo sequences of varying TE. Tendon signal-to-noise ratios (SNR) were determined for each study and images were evaluated for visual evidence of tendon infrastructure. RESULTS: Tendon SNR demonstrated a strong inverse relationship to TE at each field strength. Signal-to-noise ratio values of 10 to 40 were achieved on the shortest TE studies (TE < 3 msec). Tendon infrastructure could be visualized and was primarily dependent on in-plane resolution and to a lesser extent on SNR. CONCLUSIONS: Short echo time MR imaging enhances tendon signal. Under optimal imaging conditions, tendon infrastructure can be evaluated. As high-performance gradient coil systems become available for clinical imaging systems, the authors believe similar results will be achievable on routine diagnostic studies and foresee an expanding role for MRI in the evaluation of tendon disease.

Anatomical model of the cochlea of the alligator lizard.

The three-dimensional structure of the cochlea of the alligator lizard was examined and an anatomical model was constructed. Separate pieces of the model represent the cochlear duct and posterior branch of the eighth cranial nerve. These pieces fit together inside a transparent plastic piece that represents the bony capsule. In this paper, the method used to construct the anatomical model is described, and three-dimensional features of cochlear anatomy are illustrated.

Deformations of the isolated mouse tectorial membrane produced by oscillatory forces.

Mechanical properties of the isolated tectorial membrane (TM) of the mouse were measured by applying oscillatory shear forces to the TM with a magnetic bead (radius approximately 10 mcm). Sinusoidal forces at 10 Hz with amplitudes from 5 to 33 nN were applied tangentially to the surfaces of 11 TMs. The ratio of force to bead displacement ranged from 0.04 to 0.98 N/m (median: 0.18 N/m, interquartile range: 0.11-0.30 N/m, n=90). Increasing frequency from 10 to 100 Hz decreased the magnitude of the displacement of the magnetic bead by 6-7.3 dB/decade. The phase of the displacement lagged that of the stimulus current by approximately 27-44 degrees across frequencies. Displacement of the adjacent tissue decreased as the distance from the magnetic bead increased. Space constants were of the order of tens of micrometers. Forces with equal amplitude and frequency were applied radially and longitudinally. Longitudinal displacements in response to longitudinal forces were 1-10 times as large as radial displacements in response to radial forces in 85% of 560 paired measurements. These results suggest that the following mechanical properties of the TM are important. (1) Viscoelasticity: The frequency dependence of TM displacement lies between that of a purely viscous and a purely elastic material, suggesting that both are important. (2) Mechanical coupling: Space constants indicate that hair bundles could interact mechanically with adjacent hair bundles via the TM. (3) Anisotropy: The mechanical impedance is greater in the radial direction than it is in the longitudinal direction. This mechanical anisotropy correlates with anatomical anisotropies, such as the radially oriented fibrillar structure of the TM.

The role of fluid inertia in mechanical stimulation of hair cells.

Hair cells, which are the receptor cells of hearing and equilibrium in vertebrates, produce electrical responses when their hair bundles are displaced by sensory stimuli. This paper summarizes the results of a theoretical study of the fluid mechanics of hair-bundle motion. The principal conclusion is that fluid inertia, which has not been included in previous studies, plays a critical role in the mechanics of hair bundles and, hence, in the processes of sensory reception in hair-cell organs.

Equilibrium behavior of an isotropic polyelectrolyte gel model of the tectorial membrane: effect of pH.

An isotropic polyelectrolyte gel model (Weiss and Freeman, 1996a) of the tectorial membrane (TM) was extended to incorporate the effect of pH. The effect of pH is analyzed in order to interpret measurements of the effect of pH on the dimensions of the TM (Freeman et al., 1996a). The pH dependence of the model results from the binding of hydrogen ions to TM macromolecules--to both neutral sites with basic pK and negatively charged sites with acidic pK. Parameters of the model can be based on estimates of the concentration in the TM of collagen and glycosaminoglycans (GAGs), two identified constituents of the TM that account for approximately 40% of its dry weight. The resulting model shows swelling responses at both high and low pH that are qualitatively similar to the measurement on the TM but that differ quantitatively. Alternatively, parameters of the model can be chosen in an ad hoc fashion to closely fit the measurements of TM swelling as a function of pH. Taken together these results suggest that either estimates of the collagen and GAG content of the TM are in error or constituents of the TM, which have not yet been identified, contain appreciable pH-dependent fixed charge and contribute to the swelling behavior of the TM.

Superposition of hydrodynamic forces on a hair bundle.

Vertebrates sense sound, orientation, and motion by means of bundles of microscopic sensory hairs that protrude from the surfaces of receptor (hair) cells. To determine the effects of the sensory epithelium, tectorial structures, and fluids on the motions of hair bundles, we examine a class of mathematical models in which hair-cell organs are represented as a system of rigid mechanical structures surrounded by fluid. The epithelium and tectorial structures are represented by rigid basal and tectorial plates, respectively; the hair bundle by a rigid body hinged to the basal plate. When the displacements of these structures are small, the equations of motion for the fluid are predominately linear. Therefore, both the fluid velocity and the force of fluid origin on the body can be expressed as a sum of components; each component results from motion of a single structure while all others are stationary. This analysis leads to a network description of the motion of the rigid body in which hydrodynamic forces are segregated from mechanical forces. The separation of hydrodynamics and mechanics not only clarifies the effects of fluids on motion but also minimizes the number of hydrodynamic computations needed to analyze models of hair-bundle motion.

Hydrodynamic forces on hair bundles at low frequencies.

We have analyzed a model for the motion of hair bundles of hair cells at low frequencies. In the model, hair-cell organs are represented as a system of rigid mechanical structures surrounded by fluid. A rigid body, that represents a hair bundle, is hinged to a vibrating plate that represents the sensory epithelium. These structures are surmounted by a second vibrating plate that represents a tectorial structure. The analysis shows that both viscous and inertial properties of the fluid are important even at asymptotically low frequencies. The relative importance of these properties depends critically on the presence and mode of motion of the tectorial plate. As a result, the angular displacement of the body at low frequencies can be proportional to basal plate displacement, velocity, acceleration, or to no simple integral of its motion; the functional relation depends upon the disposition of the tectorial plate.

Hydrodynamic forces on hair bundles at high frequencies.

We have analyzed a model for the motion of hair bundles of hair cells at high frequencies. In the model, hair-cell organs are represented as a system of rigid mechanical structures surrounded by fluid. A rigid body, that represents a hair bundle, is hinged to a vibrating plate that represents the sensory epithelium. These structures are surmounted by a second vibrating plate that represents a tectorial structure. The analysis shows that at high frequencies, fluid forces cause the rigid body to move as though it were attached to the plates with a system of levers. As a result, the angular displacement of the rigid body is proportional to the displacements of the plates even when there are no mechanical attachments of the body to the tectorial plate. This result is independent of both the size and the shape of the rigid body and independent of the presence and proximity of the tectorial plate, although the constant of proportionality depends upon these factors. Therefore, the mechanical stimulation of hair cells may be particularly simple at high frequencies where the structural differences in hair bundles and tectorial attachments--that have been shown to be important at low frequencies--play a less important role.

Hydrodynamic analysis of a two-dimensional model for micromechanical resonance of free-standing hair bundles.

To investigate the role of inner ear fluids and structures on mechanical stimulation of the hair bundles of hair cells, we analyzed a two-dimensional structure that consists of: a rectangular flap (which represents a hair bundle) attached to a flat basal plate (which represents the surface of the epithelium that contains the hair cells) with a spring-loaded hinge (that represents the compliant attachment of a hair bundle to the hair cell body) and surrounded by a viscous fluid (that represents endolymph). We computed the fluid velocity as well as the forces on and motion of the flap in response to sinusoidal vibration of the plate by numerical integration of the hydrodynamic equations, and--at asymptotically low and high frequencies--by analytic methods. The results suggest that: (1) the surface of the sensory epithelium, from which hair bundles project into fluid, plays an important part in the production of fluid forces on hair bundles; (2) both fluid inertia and viscosity play a key role in hair bundle mechanics; (3) passive mechanical resonances are likely to contribute to both frequency selectivity and frequency-to-place coding in the inner ear.

The osmotic response of the isolated tectorial membrane of the chick to isosmotic solutions: effect of Na+, K+, and Ca2+ concentration.

Changes in the size, shape, and structure of the isolated tectorial membrane of the chick were measured in response to isosmotic changes in the ionic composition of the perfusion solution. Substitution of artificial perilymph (AP) for artificial endolymph (AE) caused a small (approximately 15%), slow (time constants tau approximately 12 min) shrinkage of the thickness of the tectorial membrane that was largely reversed on return to AE. Substitution of AP for AE alters not only the predominate cation (from K+ to Na+) but also the Ca2+ concentration (from < 7 mumol/l to 2 mmol/l). Additional experiments were performed to separate effects of each of these changes. When a high-Na+, low-Ca2+ solution was substituted for a high-K+, low-Ca2+ solution (AE), the tectorial membrane swelled significantly, often to more than twice its original thickness (the largest swelling was 337%), with a slow time course (tau approximately 23 min). Addition of the Ca2+ to either high-K+ or high-Na+ solutions caused rapid shrinkage of the tectorial membrane (tau approximately 2-3 min). Addition of the Ca2+ chelator EGTA caused rapid swelling (tau approximately 4 min). Large osmotic responses were only partially reversible and caused long-lasting changes. For example, long-duration solution changes that produced large, rapid osmotic responses early in an experiment tended to produce smaller and slower responses later in the experiment. In contrast, the small osmotic responses to short-duration solution changes were repeatable for tens of hours. Changes in ionic composition of the bath affected not only the thickness of the tectorial membrane but also its other dimensions. Responses were not generally isotropic; both the size and shape of the tectorial membrane generally changed. Consistent changes in microstructure accompanied the osmotic changes.

The osmotic response of the isolated, unfixed mouse tectorial membrane to isosmotic solutions: effect of Na+, K+, and Ca2+ concentration.

Changes in the size, shape, and structure of the isolated tectorial membrane (TM) of the mouse were measured in response to isosmotic changes in the ionic composition of the bathing solution. Substitution of artificial perilymph (AP) for artificial endolymph (AE) caused a small (approximately 1%) shrinkage of the TM's thickness. This substitution alters not only the predominate cation (from K+ to Na+) but also the Ca2+ concentration (from 20 mumol/l to 2 mmol/l). When the predominate cation was changed from K+ to Na+, while holding Ca2+ concentration constant, results depended on Ca2+ concentration: there was a small (approximately 1%) swelling for 20 mumol/l Ca2+, larger (approximately 14%) swelling for lower (< 7 mumol/l) concentrations of Ca2+, and little response for 2 mmol/l Ca2+ or for solutions containing the Ca2+ chelator EGTA. Addition of Ca2+ while holding the predominate cation constant caused shrinkage of the TM; both removal of Ca2+ and addition of the Ca2+ chelator EGTA caused swelling. Swelling responses were largely reversible if the magnitude of the swelling was small. Responses greater than a few percent were only partially reversible and caused long-lasting changes. Changes in ionic composition of the bath affected not only the thickness of the TM but also its other dimensions. Solution changes that increase TM thickness tend to cause radial shearing motions of the surfaces of the TM, which are accompanied by small decreases in width. Little change in length was observed. Although the responses were non-isotropic, increases in thickness were highly correlated with increases in volume. Swelling of the TM was also accompanied by a reduction in prominence of its radially oriented fibrillar structure. These results for the isolated TM of the mouse are qualitatively similar to those obtained previously for the isolated chick TM (Freeman et al., 1994) but different from those obtained for the in vitro mouse TM (Kronester-Frei, 1979a).

Effect of lymph composition on an in vitro preparation of the alligator lizard cochlea.

The effects of different artificial lymphs on the cochlear duct of the alligator lizard were studied in an in vitro preparation. The duct was dissected and cemented to the glass floor of a chamber that had been filled with an artificial lymph. The vestibular membrane was removed and latex beads (1-5 microns in diameter) were allowed to settle on the endolymphatic surface of the duct. During perfusion with an artificial lymph solution, the positions of beads were measured and video images of the duct were obtained. Artificial lymphs were isosmotic and included artificial endolymph (AE), artificial perilymph (AP), Leibovitz's L-15 culture medium, an AE solution whose calcium concentration was the same as that of AP, and AE and AP solutions in which gluconate was substituted for chloride ions. Results obtained in AE were consistently different from those in other lymphs. The displacements of beads, the projected area of the papilla, the occurrence of blebs, and direct observation of cells in the duct all indicated that the tissue swelled in AE (with or without 2 mmol/l Ca) but showed no consistent shrinking or swelling in any of the other artificial lymphs. Thus for the solutions we used, the presence of both potassium and chloride was required to elicit the swelling response to isosmotic artificial lymphs. There were some regional differences in the swelling response: the swelling of the endolymphatic surface of the tissue in a direction orthogonal to the basilar membrane surface was smaller on the free-standing region of the basilar papilla than either on the tectorial membrane or on the hyaline epithelial cells. The preparation was osmotically stable in AP and in both AE and AP solutions in which gluconate was substituted for chloride ions. After exposure to these solutions for as much as 300 min, the preparation showed no gross signs of deterioration visible with the light microscope, and continued to exhibit a highly specific osmotic response to the composition of the bathing medium.

UVB-induced ERK/AKT-dependent PTEN suppression promotes survival of epidermal keratinocytes.

Ultraviolet (UV) radiation in sunlight is the major environmental cause of skin cancer. PTEN (phosphatase and tensin homolog deleted on chromosome 10) is a proven critical tumor suppressor. We report here that UVB downregulates PTEN in primary human keratinocytes, human HaCaT keratinocytes and mouse skin. As compared with normal skin, PTEN levels are reduced in human actinic keratosis, a precancerous skin lesion caused by solar UV. PTEN downregulation is mediated by two mechanisms: (1) PTEN is cleaved by active caspase in apoptotic cells in which AKT activation is reduced; and (2) PTEN transcription is suppressed in surviving cells, and this suppression is independent of caspase activation and occurs in parallel with increased ERK and AKT activation. We report here that the combination of ERK and AKT activation is crucial for PTEN suppression in surviving cells following UVB irradiation. AKT activation is higher in UVB-irradiated surviving cells as compared with unirradiated cells. The ERK and AKT pathways are involved in sustaining PTEN suppression in UVB-exposed cells. Increasing PTEN expression enhances apoptosis of keratinocytes in response to UVB irradiation. Our findings indicate that (1) UVB radiation suppresses PTEN expression in keratinocytes; and (2) the ERK/AKT/PTEN axis may form a positive feedback loop following UVB irradiation. Our identification of PTEN as a critical molecular target of UVB provides new insights into the pathogenesis of skin cancer.

Decoupling: theory and practice. II. State of the art: in vivo applications of decoupling.

Current methods for broadband heteronuclear decoupling are reviewed from a historical perspective. The principal concern is that decoupling should be effective over a wide range of chemical shifts without undue radiofrequency heating of the sample, particularly when human patients are involved. Continuous-wave methods are the least efficient in this respect, followed by noise decoupling. Composite pulse schemes offer a more effective use of radiofrequency power, while adiabatic passage methods are the most efficient of all. Bi-level decoupling employs a low level of radiofrequency irradiation during the relaxation delay to maintain the nuclear Overhauser effect, with a higher level during signal acquisition in order to decouple over a wide frequency band. All decoupling sequences introduce cycling sidebands into the observed spectrum, and schemes are described to minimize the intensity of these artifacts. In part II, practical applications of decoupling methods are examined in the context of in vivo spectroscopy, where the improvements in sensitivity and resolution through broadband decoupling can be critical for solving clinical problems. Attention is focused on the regulatory limits on power deposition in these experiments. A tabulation of the existing work on decoupling in biological tissue is presented, mainly involving 31P and 13C spectroscopy in vivo or in vitro.