Insulin-like growth factor-1 regulates the mechanosensitivity of chondrocytes by modulating TRPV4.

Both mechanical and biochemical stimulation are required for maintaining the integrity of articular cartilage. However, chondrocytes respond differently to mechanical stimuli in osteoarthritic cartilage when biochemical signaling pathways, such as Insulin-like Growth Factor-1 (IGF-1), are altered. The Transient Receptor Potential Vanilloid 4 (TRPV4) channel is central to chondrocyte mechanotransduction and regulation of cartilage homeostasis. Here, we propose that changes in IGF-1 can modulate TRPV4 channel activity. We demonstrate that physiologic levels of IGF-1 suppress hypotonic-induced TRPV4 currents and intracellular calcium flux by increasing apparent cell stiffness that correlates with actin stress fiber formation. Disruption of F-actin following IGF-1 treatment results in the return of the intracellular calcium response to hypotonic swelling. Using point mutations of the TRPV4 channel at the microtubule-associated protein 7 (MAP-7) site shows that regulation of TRPV4 by actin is mediated via the interaction of actin with the MAP-7 domain of TRPV4. We further highlight that ATP release, a down-stream response to mechanical stimulation in chondrocytes, is mediated by TRPV4 during hypotonic challenge. This response is significantly abrogated with IGF-1 treatment. As chondrocyte mechanosensitivity is greatly altered during osteoarthritis progression, IGF-1 presents as a promising candidate for prevention and treatment of articular cartilage damage.

American Dreaming: Really Reading The Great Gatsby.

F. Scott Fitzgerald's The Great Gatsby (1925) is one of the best known and most widely read and taught novels in American literature. It is so familiar that even those who have not read it believe that they have and take for granted that they know about its main character and theme of the American Dream. We need to approach The Great Gatsby as if it were new and really read it, paying close attention to Fitzgerald's literary language. His novel gives us a vivid depiction of and insight into income inequality as it existed in the 1920s and, by extension, as it exists today, when the American Dream is even more limited to the fortunate few, not within reach of the many. When we really read The Great Gatsby, we perceive and understand the American dimension of the novel and appreciate, too, the global range and relevance that in it Fitzgerald has achieved. It is a great American book and a great book of world literature.

An algorithm for 353 odor detection thresholds in humans.

One hundred and ninety three odor detection thresholds, ODTs, obtained by Nagata using the Japanese triangular bag method can be correlated as log (1/ODT) by a linear equation with R(2) = 0.748 and a standard deviation, SD, of 0.830 log units; the latter may be compared with our estimate of 0.66 log units for the self-consistency of Nagata's data. Aldehydes, acids, unsaturated esters, and mercaptans were included in the equation through indicator variables that took into account the higher potency of these compounds. The ODTs obtained by Cometto-Muñiz and Cain, by Cometto-Muñiz and Abraham, and by Hellman and Small could be put on the same scale as those of Nagata to yield a linear equation for 353 ODTs with R(2) = 0.759 and SD = 0.819 log units. The compound descriptors are available for several thousand compounds, and can be calculated from structure, so that further ODT values on the Nagata scale can be predicted for a host of volatile or semivolatile compounds.

Human chemosensory perception of methyl isothiocyanate: chemesthesis and odor.

An unpublished laboratory study by Russell and Rush (1996) showed that human subjects sense the presence of methyl isothiocyanate (MITC) via the eyes at concentrations as low as hundreds of ppb in air, with dependence upon duration of exposure. The longer the stimulation, the lower the concentrations sensed. Application of benchmark concentration (BMC10) modeling indicated a best estimate of 330 ppb by the end of 4h. With a confidence limit (BMCL) applied, the level dropped to 220 ppb, when employing a probit model. Receptors known as TRPA1 ion channels present in trigeminal and associated peripheral afferent nerves have shown particular sensitivity to isothiocyanates. Sensitivity to these electrophiles, which occur naturally in plants (e.g., capers and mustard greens), most likely derives from a mechanism of reversible covalent bonding. Such sensing can provide warning of potential damage rather than actual damage itself. Based upon its reputation as a lachrymator, Russell and Rush assumed that the eyes would sense MITC, before the upper airways, so gathered no data from the airways, except for odor. Field results from spills and results of acute exposures to animals covered in Dourson et al. (2010) add pertinent information on the matter.

An AP-3-dependent mechanism drives synaptic-like microvesicle biogenesis in pancreatic islet beta-cells.

Pancreatic islet beta-cells contain synaptic-like microvesicles (SLMVs). The origin, trafficking, and role of these SLMVs are poorly understood. In neurons, synaptic vesicle (SV) biogenesis is mediated by two different cytosolic adaptor protein complexes, a ubiquitous AP-2 complex and the neuron-specific AP-3B complex. Mice lacking AP-3B subunits exhibit impaired GABAergic (inhibitory) neurotransmission and reduced neuronal vesicular GABA transporter (VGAT) content. Since beta-cell maturation and exocytotic function seem to parallel that of the inhibitory synapse, we predicted that AP-3B-associated vesicles would be present in beta-cells. Here, we test the hypothesis that AP-3B is expressed in islets and mediates beta-cell SLMV biogenesis. A secondary aim was to test whether the sedimentation properties of INS-1 beta-cell microvesicles are identical to those of bona fide SLMVs isolated from PC12 cells. Our results show that the two neuron-specific AP-3 subunits beta3B and mu3B are expressed in beta-cells, the first time these proteins have been found to be expressed outside the nervous system. We found that beta-cell SLMVs share the same sedimentation properties as PC12 SLMVs and contain SV proteins that sort specifically to AP-3B-associated vesicles in the brain. Brefeldin A, a drug that interferes with AP-3-mediated SV biogenesis, inhibits the delivery of AP-3 cargoes to beta-cell SLMVs. Consistent with a role for AP-3 in the biogenesis of GABAergic SLMV in beta-cells, INS-1 cell VGAT content decreases upon inhibition of AP-3 delta-subunit expression. Our findings suggest that beta-cells and neurons share molecules and mechanisms important for mediating the neuron-specific membrane trafficking pathways that underlie synaptic vesicle formation.

Making scents: dynamic olfactometry for threshold measurement.

Data on human odor thresholds show disparities huge enough to marginalize olfactory psychophysics and delegitimize importation of its data into other areas. Variation of orders of magnitude from study to study, much of it systematic, threatens meaningful comparisons with animal species, comparison between in vivo with in vitro studies, the search for molecular determinants of potency, and use of olfactory information for environmental or public health policy. On the premise that good experimental results will flow from use of good tools, this report describes a vapor delivery system and its peripherals that instantiate good tools. The vapor delivery device 8 (VDD8) provides flexibility in range of delivered concentrations, offers definable stability of delivery, accommodates solvent-free delivery below a part per trillion, gives a realistic interface with subjects, has accessible and replaceable components, and adapts to a variety of psychophysical methodologies. The device serves most often for measurement of absolute sensitivity, where its design encourages collection of thousands of judgments per day from subjects tested simultaneously. The results have shown humans to be more sensitive and less variable than has previous testing. The VDD8 can also serve for measurement of differential sensitivity, discrimination of quality, and perception of mixtures and masking. The exposition seeks to transmit general lessons while it proffers some specifics of design to reproduce features of the device in a new or existing system. The principles can apply to devices for animal testing.

The biological and toxicological activity of gases and vapors.

A large amount of data on the biological and toxicological activity of gases and vapors has been collected from the literature. Processes include sensory irritation thresholds, the Alarie mouse test, inhalation anesthesia, etc. It is shown that a single equation using only five descriptors (properties of the gases and vapors) plus a set of indicator variables for the given processes can correlate 643 biological and non-lethal toxicological activities of 'non-reactive' compounds with a standard deviation of 0.36 log unit. The equation is scaled to sensory irritation thresholds obtained by the procedure of Cometto-Muñiz, and Cain provides a general equation for the prediction of sensory irritation thresholds in man. It is suggested that differences in biological/toxicological activity arise primarily from transport from the gas phase to a receptor phase or area, except for odor detection thresholds where interaction with a receptor(s) is important.

Concentration-detection functions for the odor of homologous n-acetate esters.

Using air-dilution olfactometry, we measured concentration-response functions for the odor detection of the homologous esters ethyl, butyl, hexyl, and octyl acetate. Stimuli were delivered by means of an 8-station vapor delivery device (VDD-8) specifically designed to capture odor detection performance by humans under environmentally realistic conditions. Groups of 16-17 (half female) normosmic (i.e., having a normal olfaction) non-smokers (ages 18-38) were tested intensively. The method involved a three-alternative forced-choice procedure against carbon-filtered air, with an ascending concentration approach. Delivered concentrations were confirmed by gas chromatography before and during actual testing. A sigmoid (logistic) model provided an excellent fit to the odor detection functions both at the group and individual levels. Odor detection thresholds (ODTs) (defined as the half-way point between chance and perfect detection) decreased from ethyl (245 ppb by volume), to butyl (4.3 ppb), to hexyl acetate (2.9 ppb), and increased for octyl acetate (20 ppb). Interindividual threshold variability was near one and always below two orders of magnitude. The steepness of the functions increased slightly but significantly with carbon chain length. The outcome showed that the present thresholds lie at the very low end of those previously reported, but share with them a similar relative trend across n-acetates. On this basis, we suggest that a recent quantitative structure-activity relationship (QSAR) for ODTs can be applied to these and additional optimized data, and used to describe and predict not just ODTs but the complete underlying psychometric odor functions.

Solvation parameters for mercury and mercury(II) compounds: calculation of properties of environmental interest.

Descriptors have been determined for four inorganic mercury(II) species and for seventeen organic mercury(II) species, using experimental literature data. These descriptors can then be used in equations that we have already set out in order to estimate a large number of physicochemical properties. These include the water to octanol partition coefficient and the gas to water partition coefficient. For the organic mercury(II) species, including dimethylmercury and the methylmercury(II) halides, the latter has been estimated over the temperature range 273-373 K.

Chemesthetic responses to airborne mineral dusts: boric acid compared to alkaline materials.

OBJECTIVES: (1) To assess the relation between occupationally relevant exposures to dust of boric acid and magnitude of feel in the eye, nose, and throat during activity (pedaling) equal to light industrial work. (2) To compare feel from the dust of boric acid with that of the alkaline dusts calcium oxide and sodium tetraborate pentahydrate (sodium borate). (3) To chart how magnitude of feel changes with time in exposures up to 3/4 h. METHODS: Twelve subjects, six males and six females, participated in duplicate sessions of exposure to 2.5, 5, and 10 mg m(-3) of boric acid, 10 mg m(-3) of sodium borate, 2.5 mg m(-3) of calcium oxide presented as calcium oxide alone or diluted with hydrated calcium sulfate, and 0 mg m(-3) (blank). Exposures occurred in a plastic dome suspended over the head and closed around the neck with rubber dam. Measurements pre- and post-exposure included nasal secretion and nasal resistance. Measurements during exposure included rated magnitude of feel in the eye, nose, and throat, and respiration (Respitrace System). Six concentrations of carbon dioxide ranging from just below detectable to sharply stinging gave subjects references for their ratings. RESULTS: In general, feel increased for periods up to half an hour, then either declined or held at a plateau. Each material had a temporal signature. The nose led with the highest feel, followed by the throat, then the eyes. This hierarchy proved weakest for boric acid; at one level of exposure, magnitude in the throat overtook that in the nose. Accompanying measures implied that change of feel with time occurred neither because of an increase in dilution of the dissolved dusts in newly secreted mucus nor an increase of consequence in nasal resistance. Most likely, sensory adaptation determined the change. Boric acid of 10 mg m(-3) fell slightly and insignificantly below 10 mg m(-3) sodium borate in feel. Boric acid, though, showed a relatively flat dose-response relationship, i.e., a change in level caused little change in feel. CONCLUSIONS: The time-constant for feel from dusts lies on the order of tens of minutes. A flat concentration-response function for boric acid and a notable response from the throat suggests that perceived dryness, not mediated by acidity but perhaps by osmotic pressure, may account for the feel evoked at levels of exposure at or below 10 mg m(-3). More acidic dusts that could actually change nasal pH may trigger sensations differently.

A quantitative structure activity analysis on the relative sensitivity of the olfactory and the nasal trigeminal chemosensory systems.

We have applied a quantitative structure-activity relationship (QSAR) approach to analyze the chemical parameters that determine the relative sensitivity of olfaction and nasal chemesthesis to a common set of volatile organic compounds (VOCs). We used previously reported data on odor detection thresholds (ODTs) and nasal pungency thresholds (NPTs) from 64 VOCs belonging to 7 chemical series (acetate esters, carboxylic acids, alcohols, aliphatic aldehydes, alkylbenzenes, ketones, and terpenes). The analysis tested whether NPTs could be used to separate out "selective" chemosensory effects (i.e., those resting on the transfer of VOCs from the gas phase to the receptor phase) from "specific" chemosensory effects in ODTs. Previous work showed that selective effects overwhelmingly dominate chemesthetic potency whereas both selective and specific effects control olfactory potency. We conclude that it is indeed possible to use NPTs to separate out selective from specific effects in ODTs. Among the series studied, aldehydes and acids, except for formic acid, show clear specific effects in their olfactory potency. Furthermore, for VOCs whose odor potency rests mainly on selective effects, we have developed a QSAR equation that can predict their ODTs based on their NPTs.

Concentration-detection functions for eye irritation evoked by homologous n-alcohols and acetates approaching a cut-off point.

We measured the concentration-detection (i.e., psychometric) functions for the eye irritation evoked by three homologous n-alcohols (1-nonanol, 1-decanol and 1-undecanol) and two homologous acetates (nonyl and decyl acetate). A vapor delivery device based on a dynamic dilution of stimuli in nitrogen served to present various concentrations of each compound, including the undiluted vapor, to the subjects (n >or= 26). Delivered concentrations were quantified by gas chromatography. Detection probability (P) was assessed via a three-alternative, forced-choice procedure and quantified on a scale ranging from P = 0.0 (chance detection) to P = 1.0 (perfect detection). Flow rate to the eye equaled 2.5 l/min and time of exposure was 6 s. The functions for 1-undecanol and decyl acetate plateaued at P approximately 0.5 and P approximately 0.25, respectively, such that further increases in concentration failed to increase detection notably. Thus, both series reached a break point, or cut-off, in the detection of ocular irritation. The present outcome provides additional evidence that the cut-off does not rest on the low vapor concentration of the homolog but, more likely, on the homolog exceeding a critical molecular dimension(s), which prevents it from interacting effectively with the appropriate receptors.

Odor and chemesthesis from exposures to glutaraldehyde vapor.

OBJECTIVES: Assessment of olfactory and chemesthetic sensitivity (feel, sensory irritation) to vapor of glutaraldehyde in young adult females. For chemesthetic sensitivity, assessment included the variable of duration, with focus on whether concentrations initially too low to evoke feel in the eye or upper airway might do so in exposures up to 15 min. METHODS: Experiment 1 probed sensitivity with forced-choice testing of detection over ranges of concentrations appropriate to three endpoints: odor, feel in the eye, and feel in the nose. A subject participated in hours of testing per endpoint to yield enough data to erect a psychometric (concentration-response) function. Exposure in Experiment 1 entailed use of a vapor-delivery system that stimulated sites of interest separately. Exposure in Experiment 2 occurred in the ambient environment of a chamber, with the sites stimulated simultaneously. In that case, subjects rated confidence by the minute that they felt the presence of vapor in the eyes, nose, and throat during exposures of 15 minutes to 35, 50, 75, and 100 ppb, a blank, and an odor control of mild heptane. RESULTS: In Experiment 1, the typical subject achieved 50% detection (threshold) of odor at 0.3 ppb. The typical subject achieved 50% detection of feel in the eye and nose at 390 and 470 ppb, respectively. Psychometric functions for feel showed much sharper dependence on concentration than those for odor. In Experiment 2, confidence in detection of feel migrated progressively away from no-with certainty toward the zone of uncertainty, with bigger change when the exposures contained any glutaraldehyde. The ratings of confidence failed, however, to show distinguish among these concentrations. CONCLUSIONS: Glutaraldehyde has much higher odor potency than previously thought. Its green-apple odor should signal presence of the vapor at levels more than a 100-fold below any that might evoke sensory irritation in brief exposures. Exposures that start decidedly below irritating (100 ppb and below) seem unlikely to turn irritating over time. Although the effects from these concentrations differentiated themselves from those of air and an odor control, they exhibited none of the concentration dependence seen for sensations of feel. They seemed likely driven by the penetrating odor of glutaraldehyde.

Chemesthesis from volatile organic compounds: Psychophysical and neural responses.

In Experiment 1, subjects sought to localize the nostril stimulated, left or right, in tests with nine esters (acetates, propionates, and butyrates) at concentrations meant to trigger chemesthesis (pungency, irritation). The task produced psychometric functions for chemesthetic detection unconfounded by olfactory sensations. The functions indicated a sharp transition from no detection to perfect detection, rather uniform across the esters, which themselves varied in potency by two log units. The correlation between the thresholds for the eight materials that yielded thresholds and predictions from a published linear free energy relationship (LFER) equaled 0.99. In Experiment 2, amplitude of the negative mucosal potential (NMP) was recorded from the septum. The resulting functions also increased with concentration sharply. Against a criterion amplitude of the NMP, thresholds measured in the first experiment (and predictions from the LFER) correlated 0.99. The NMP seems to offer an adequate objective measure of sensory irritation. The LFER, although effective predictively, could stand to have a parameter to anticipate that molecules beyond a certain size fail to trigger irritation. In the present case, a cut-off of chemesthetic potency occurred between butyl butyrate and hexyl butyrate for the group of subjects, with some variation of the boundary among individuals.

Chemical boundaries for detection of eye irritation in humans from homologous vapors.

In a series of experiments, we looked at a "cutoff" effect for the detection of eye irritation from neat vapors of homologous n-alkylbenzenes and 2-ketones. Stimuli comprised pentyl, hexyl, and heptyl benzene, 2-dodecanone, and 2-tridecanone, presented to each eye at 4 and 8 l/min for 6 sec, using a three-alternative forced-choice procedure against blanks. Detection probability corrected for chance (i.e., detectability) decreased with carbon chain length such that heptyl benzene and 2-tridecanone were virtually undetectable, irrespective of flow rate to the eye. Heating both stimuli sources to 37 degrees C (body temperature) from 23 degrees C (room temperature) increased vapor concentration by 5.0 and 6.9 times, respectively, for heptyl benzene and 2-tridecanone. Still, both chemicals failed to show increased detection for 13 of the 21 participants. In addition, plots of experimentally measured and calculated eye irritation thresholds as a function of carbon chain length for each series indicated that, based on the trend, the concentration of the two cutoff homologs at 37 degrees C should have been high enough to allow detection. Taken together, the results suggest that these cutoffs rest on limitations related to the dimension of the molecules rather than on limitations related to their vapor concentration. For example, the stimulus molecule could exceed the size that allows it to fit into the receptor pocket of a receptive protein. Plots of calculated molecular dimensions across homologous alkylbenzenes, from ethyl to dodecylbenzene, and across 2-ketones, from 2-octanone to 2-octadecanone, provided additional support to the above conclusion.

Production of monoclonal antibodies against Prox1.

Prox1 is a divergent homeodomain protein important for the development of the lens, retina, liver, pancreas, and lymphatic vasculature. Prox1 expression is highly upregulated in transformed hepatocytes and has been used as a marker to distinguish lymphatic from blood vasculature. We produced recombinant human Prox1 (amino acids 547-737) fused to glutathione S-transferase (GST) and used it to create two hybridomas, 5G10 and 4G10. Both of these hybridomas produced monoclonal antibodies able to detect Prox1 by immunofluorescence in lenses from diverse terrestrial vertebrates, including humans, rats, chickens, and lizards, although 5G10 was generally more sensitive in this application. Further, 4G10 was able to robustly detect endogenous and recombinant Prox1 in both cell and tissue extracts by Western blotting, while 5G10 was notably less sensitive for this purpose. These monoclonal antibodies will be useful for diverse studies on the role of Prox1 in both normal development and disease processes in terrestrial vertebrates.