Reduction in TRPC4 expression specifically attenuates G-protein coupled receptor-stimulated increases in intracellular calcium in human myometrial cells

Canonical transient receptor potential (TRPC) proteins may play a role in regulating changes in intracellular calcium ([Ca²⁺]_i). Human myometrium expresses TRPC4, TRPC1 and TRPC6 mRNAs in greatest relative abundance. Contributions of TRPC4 to increases in [Ca²⁺]_i were assessed in PHM1–41 and primary human uterine smooth muscle (UtSMC) cells using short hairpin RNAs (shRNAs). Based on a reporter assay screen, one shRNA was selected to construct an adenoviral expression vector (TC4sh1). TC4sh1 induced both mRNA and protein TRPC4 knockdown in PHM1–41 cells without affecting expression of other TRPCs. Signal-regulated Ca²⁺ entry (SRCE), defined as a stimulus- and extracellular Ca²⁺-dependent increase in [Ca²⁺]_i, was measured in PHM1–41 cells treated with oxytocin (G-protein coupled receptor (GPCR)-stimulated), thapsigargin (store depletion-stimulated), and OAG (diacylglycerol-stimulated), using Fura-2. Cells infected with TC4sh1 exhibited attenuated oxytocin-, ATP- and PGF2α-mediated SRCE, but no change in thapsigargin- or OAG-stimulated SRCE. Similar results were obtained in primary uterine smooth muscle cells. Additionally, cells expressing TC4sh1 exhibited a significantly smaller increase in channel activity in response to oxytocin administration than did cells infected with empty virus. These data show that, in human myometrial cells, knockdown of endogenous TRPC4 specifically attenuates GPCR-stimulated, but not thapsigargin- or OAG-stimulated extracellular calcium-dependent increases in [Ca²⁺]_i. These data imply that, in this cellular context, the mechanisms regulating extracellular Ca²⁺-dependent increases in [Ca²⁺]_i are differentially affected by different signaling pathways.     

Habituation and desensitization as methods for reducing fearful behavior in singly housed rhesus macaques

Operant conditioning using positive reinforcement techniques has been used extensively in the management of nonhuman primates in both zoological and laboratory settings. This research project was intended to test the usefulness of counter-conditioning techniques in reducing the fear-responses of singly housed male rhesus macaques living in the laboratory environment. A total of 18 male rhesus macaques (Macaca mulatta) were selected for this project and randomly assigned to one of three groups: a desensitization training group, a husbandry training group, or a control group. Behavioral data were collected before and after a 6 weeks training and/or habituation period during which the first two groups received a total of 125 min of positive reinforcement training (and also were assumed to undergo habituation to the environment) and the control group experienced only simple habituation to the environment. Based on a Wilcoxon Matched-Pairs Sign Test, we found that a significant proportion of animals exposed to desensitization training showed a reduction in the rate at which they engaged in cringing toward humans (exact significance=0.016, one-tailed, N-ties=6), cringing in general (exact significance=0.016, one-tailed, N-ties=6), and in stress-related behaviors (exact significance=0.016, one-tailed, N-ties=6). This was not the case for animals exposed to basic husbandry training or animals in the control group. A significant proportion of desensitization-exposed animals also showed a reduction in the duration of time spent cringing toward humans (exact significance=0.016, one-tailed, N-ties=6), but not in cringing behaviors in general or in stress-related behaviors. There were not a significant proportion of animals in either the husbandry training group or the control group that showed a decrease in duration of these behaviors. Results of this study could enhance both laboratory animal welfare and laboratory animal research, and could be a first step in developing techniques for reducing fearful behavior in rhesus monkeys in the laboratory environment.     

Detecting substrate engagement: responses of tarsal campaniform sensilla in cockroaches

Sensory signals of contact and engagement with the substrate are important in the control and adaptation of posture and locomotion. We characterized responses of campaniform sensilla, receptors that encode forces as cuticular strains, in the tarsi (feet) of cockroaches using neurophysiological techniques and digital imaging. A campaniform sensillum on the fourth tarsal segment was readily identified by its large action potential in nerve recordings. The receptor discharged to contractions of the retractor unguis muscle, which engages the pretarsus (claws and arolium) with the substrate. We mimicked the effects of muscle contractions by applying displacements to the retractor apodeme (tendon). Sensillum firing did not occur to unopposed movements, but followed engagement of the claws with an object. Vector analysis of forces suggested that resisted muscle contractions produce counterforces that axially compress the tarsal segments. Close joint packing of tarsal segments was clearly observed following claw engagement. Physiological experiments showed that the sensillum responded vigorously to axial forces applied directly to the distal tarsus. Discharges of tarsal campaniform sensilla could effectively signal active substrate engagement when the pretarsal claws and arolium are used to grip the substrate in climbing, traversing irregular terrains or walking on inverted surfaces.     

A novel approach to in vivo mitral valve stress analysis

Three-dimensional (3-D) echocardiography allows the generation of anatomically correct and time-resolved geometric mitral valve (MV) models. However, as imaged in vivo, the MV assumes its systolic geometric configuration only when loaded. Customarily, finite element analysis (FEA) is used to predict material stress and strain fields rendered by applying a load on an initially unloaded model. Therefore, this study endeavors to provide a framework for the application of in vivo MV geometry and FEA to MV physiology, pathophysiology, and surgical repair. We hypothesize that in vivo MV geometry can be reasonably used as a surrogate for the unloaded valve in computational (FEA) simulations, yielding reasonable and meaningful stress and strain magnitudes and distributions. Three experiments were undertaken to demonstrate that the MV leaflets are relatively nondeformed during systolic loading: 1) leaflet strain in vivo was measured using sonomicrometry in an ovine model, 2) hybrid models of normal human MVs as constructed using transesophageal real-time 3-D echocardiography (rt-3DE) were repeatedly loaded using FEA, and 3) serial rt-3DE images of normal human MVs were used to construct models at end diastole and end isovolumic contraction to detect any deformation during isovolumic contraction. The average linear strain associated with isovolumic contraction was 0.02 ± 0.01, measured in vivo with sonomicrometry. Repeated loading of the hybrid normal human MV demonstrated little change in stress or geometry: peak von Mises stress changed by <4% at all locations on the anterior and posterior leaflets. Finally, the in vivo human MV deformed minimally during isovolumic contraction, as measured by the mean absolute difference calculated over the surfaces of both leaflets between serial MV models: 0.53 ± 0.19 mm. FEA modeling of MV models derived from in vivo high-resolution truly 3-D imaging is reasonable and useful for stress prediction in MV pathologies and repairs.     

Variation for phenotypic plasticity among populations of an invasive exotic grass

Phenotypic plasticity is a common feature of plant invaders, but little is known about variation in plasticity among invading populations. Variation in plasticity of ecologically important traits could facilitate the evolution of greater plasticity and invasiveness. We examined plasticity among invasive populations of Microstegium vimineum (Japanese stiltgrass), a widespread and often dominant grass of forests in the eastern U.S. with two separate experiments. First, we exposed seven Microstegium populations to a drought treatment in growth chambers and monitored growth and physiological responses. Then, we established a greenhouse experiment using a subset of the populations; two that exhibited the most divergent responses and one intermediate population. In the greenhouse, we manipulated drought and shade and evaluated biomass production and specific leaf area (SLA). Microstegium exhibited plasticity for biomass production and SLA in the greenhouse experiment, and populations significantly varied in the degree of plasticity under drought and shade treatments. Two populations significantly increased biomass production under favorable conditions, unlike the third population. The most productive populations also responded to shade stress via greater SLA, possibly allowing for greater utilization of available light, while the third population did not. These results show that Microstegium can exhibit plastic responses to environmental conditions. Moreover, variation for plasticity among populations provides the potential for further evolution of plasticity. Future studies should focus on the relative importance of plasticity for the success of Microstegium and other plant invaders and evaluate post-introduction evolution of plasticity.     

Chemical and histochemical studies of normal and diseased human gastrointestinal tract. I. A comparison between histologically normal colon, colonic tumours, ulcerative colitis and diverticular disease of the colon

Summary Chemical and histochemical methods were used to compare the epithelial glycoproteins from formalin-fixed surgical specimens of normal human large intestine, colonic tumours, ulcerative colitis and diverticular disease. All the epithelial glycoproteins contained fucose, galactose, glucosamine, galactosamine and, in addition, sialic acids both with and withoutO-acyl substituents in the side chain and/or at position C₄. The glycoproteins of the normal ascending and descending colons differed significantly with respect to the percentage of the sialic acids released following digestion of the de-O-acylated glycoprotein withVibrio cholera neuraminidase and to the molar fucose-sialic acid ratio. Statistical analysis of the chemical data showed that (a) compared to normal, the sialic acids of the tumour and ulcerative colitis glycoproteins from the descending colon were significantly less substituted in the side chain and at position C₄; (b) theO-acetyl substitution pattern of the sialic acids of the ulcerative colitis glycoproteins from the ascending colon and the quantitative composition of the carbohydrate prosthetic groups of the ulcerative colitis glycoproteins from both ascending and descending colons differed from normal; (c) it was not always possible to distinguish between the ulcerative colitis and tumour glycoproteins on the basis of theO-acetyl substitution pattern of their sialic acids; and (d), there were minor differences between normal glycoproteins and those from cases of diverticular disease.