The use of yeast to understand TRP-channel mechanosensitivity

Mechanosensitive (MS) ion channels likely underlie myriad force-sensing processes, from basic osmotic regulation to specified sensations of animal hearing and touch. Albeit important, the molecular identities of many eukaryotic MS channels remain elusive, let alone their working mechanisms. This is in stark contrast to our advanced knowledge on voltage- or ligand-sensitive channels. Several members of transient receptor potential (TRP) ion channel family have been implicated to function in mechanosensation and are recognized as promising candidate MS channels. The yeast TRP homolog, TRPY1, is clearly a first-line force transducer. It can be activated by hypertonic shock in vivo and by membrane stretch force in excised patches under patch clamp, making it a useful model for understanding TRP channel mechanosensitivity in general. TRPY1 offers two additional research advantages: (1) It has a large (∼300 pS) unitary conductance and therefore a favorable S/N ratio. (2) Budding yeast allows convenient and efficient genetic and molecular manipulations. In this review, we focus on the current research of TRPY1 and discuss its prospect. We also describe the use of yeast as a system to express and characterize animal TRP channels.     

Pressure-sensitive ion channel in Escherichia coli.

We have used the patch-clamp electrical recording technique on giant spheroplasts of Escherichia coli and have discovered pressure-activated ion channels. The channels have the following properties: activation by slight positive or negative pressure; voltage dependence; large conductance; selectivity for anions over cations; dependence of activity on the species of permeant ions. We believe that these channels may be involved in bacterial osmoregulation and osmotaxis.     

Use of monoclonal antibodies as sensitive and specific probes for biologically active human gamma-interferon.

Mouse monoclonal antibodies B1 and B3 are specific for natural and Escherichia coli-derived recombinant human gamma-interferon (IFN-gamma). The two antibodies recognize different epitopes of the IFN-gamma molecule and do not compete with each other's binding. We have used these two antibodies to construct a solid-phase, sandwich immunoradiometric assay for human IFN-gamma. Purified antibody B1 was coated on polystyrene beads (0.64 cm in diameter) and used as the solid-phase immunoadsorbent and antibody B3 was labeled with 125I and used as tracer. This assay can be completed in about 4 hr and is capable of detecting IFN-gamma levels in human serum or tissue culture fluids as low as 0.1 NIH reference unit/ml. Recombinant human IFN-gamma derived from E. coli was detectable at a concentration of 0.02 ng/ml. The assay appears to be specific for the biologically active forms of IFN-gamma, since after exposure to pH 2, 37 degrees C, or 56 degrees C, biological activity and reactivity in the immunoradiometric assay decreased in parallel. The immunoradiometric assay can be employed for the analysis of the structural characteristics of the human IFN-gamma molecule.     

Goitrogenesis during pregnancy and neonatal hypothyroxinaemia in a borderline iodine sufficient area

OBJECTIVE: Severe iodine deficiency disorders (IDDs) may have been eradicated in many parts of the world, but milder forms still exist and may escape detection. We evaluated the impact of pregnancy on the maternal and fetal thyroid axis in Hong Kong, a coastal city in southern China with borderline iodine intake. DESIGN: A prospective study performed in a maternity hospital. PATIENTS: Two hundred and thirty pregnant women were prospectively studied and their neonates assessed at birth. MEASUREMENTS: Urine iodine concentration, thyroid function tests and thyroid volume (TV) by ultrasound were determined in the mothers during pregnancy and up to 3 months postpartum and in the neonates. RESULTS: Increased urinary iodine concentration was seen from first trimester onwards and the proportion of women having urine iodine concentration of < 0.4 micromol/l decreased from 11.3% in the first trimester to 4.7% in the third trimester. There was progressive reduction in circulating fT4 and fT3 concentrations and free thyroxine index (FTI) with increasing gestation and the percentage of women having subnormal levels at term were 53.2%, 61.1% and 4.8%, respectively. The serum TSH concentration during pregnancy doubled towards term. In the first trimester, multiparous women had significantly larger TV than the nulliparous women (P < 0.001). By the third trimester, TV had increased by 30% (range 3-230%) so that the goitre incidence was 14.1%, 21.8%, 25.9% during the three trimesters of pregnancy, and 24.3% and 21.9% at 6 weeks and 3 months postpartum (ANOVA, P < 0.05). The change in thyroid volume during pregnancy correlated positively with the change in thyroglobulin (r = 0.225, P < 0.002) and negatively with urinary iodine concentration (r = - 0.149, P < 0.02). Fourteen women with excessive thyroidal stimulation in the second trimester (defined as those with thyroglobulin (Tg) concentrations in the highest tertile and FTI in the lowest tertile) were found to have lower urine iodine concentrations and larger TV (both P < 0.005) throughout pregnancy, and their neonates had higher cord TSH (P < 0.05), Tg (P < 0.05) and slightly larger TV (P = 0.06) as compared to the findings in 216 pregnant women without evidence of thyroid stimulation. Seven neonates (50%) born to these women had subnormal fT4 levels at birth. CONCLUSION: In a borderline iodine sufficient area, pregnancy posed an important stress resulting in higher rates of maternal goitrogenesis as well as neonatal hypothyroxinaemia and hyperthyro- trophinaemia. An adequate iodization program is necessary to eliminate iodine deficiency disorders during pregnancy.     

Alcohol Modulation of Human Normal T-Cell Activation, Maturation, and Migration

We are interested in the characterization of the effects of alcohol on human T-cell activation, maturation, and migration, because this cell population is crucial in the initiation, regulation, and propagation of cellular immunity. We and others have described the effects of both acute and chronic exposure of human immune cells to ethanol (EtOH) in vitro. Herein, we briefly, review these reports and expand this body of literature with the inclusion of new data recently obtained in our laboratory. We confirm the blunting effects of EtOH on the production of interleukin-2 and mitogen proliferative response following T-cell mitogen stimulation, and on the expression of membrane markers of activation. We show that EtOH significantly alters the expression of the CD4 cell-associated marker of activation, CD26. We report the effect of EtOH on the expression of the homing receptor CD62L by CD4⁺ cells, and on their ability to adhere by a CD18-mediated process to a defined cellular substratum. Furthermore, we demonstrate the effects of EtOH and EtOH and 0-endor-phin pretreatment on the activation of CD4⁺ lymphocytes endowed with the homing receptor CD62L.     

Effects of diallyl trisulfide on induction of apoptotic death in murine leukemia WEHI‐3 cells in vitro and alterations of the immune responses in normal and leukemic mice in vivo

Diallyl trisulfide (DATS), a chemopreventive dietary constituent and extracted from garlic, has been shown to against cultured many types of human cancer cell liens but the fate of apoptosis in murine leukemia cells in vitro and immune responses in leukemic mice remain elusive. Herein, we clarified the actions of DATS on growth inhibition of murine leukemia WEHI‐3 cells in vitro and used WEHI‐3 cells to generate leukemic mice in vivo, following to investigate the effects of DATS in animal model. In in vitro study, DATS induced apoptosis of WEHI‐3 cells through the G0/G1 phase arrest and induction of caspase‐3 activation. In in vivo study DATS decreased the weight of spleen of leukemia mice but did not affect the spleen weight of normal mice. DATS promoted the immune responses such as promotions of the macrophage phagocytosis and NK cell activities in WEHI‐3 leukemic and normal mice. However, DATS only promotes NK cell activities in normal mice. DATS increases the surface markers of CD11b and Mac‐3 in leukemia mice but only promoted CD3 in normal mice. In conclusion, the present study indicates that DATS induces cell death through induction of apoptosis in mice leukemia WHEI‐3 cells. DATS also promotes immune responses in leukemia and normal mice in vivo. © 2014 Wiley Periodicals, Inc. Environ Toxicol 30: 1343–1353, 2015.     

Life events, daily stresses and coping in patients with Graves' disease

OBJECTIVE The contribution of stress to the aetiology of Graves' disease (GD) remains controversial. We have therefore examined life events, dally stress and coping in patients with this disease. We wished to determine whether the clinical presentation of Graves' hyperthyroidism is associated with preceding stressful events. DESIGN A prospective controlled study. Ninety-five patients with newly diagnosed GD were compared to matched controls. METHOD A self-reporting questionnaire recalling life events, dally stress and coping In the twelve months preceding the diagnosis. RESULT More Graves' disease patients than controls reported negative events (P<0·0005), whereas the number of subjects reporting positive events and neutral events were similar in both groups. Graves' disease patients also experienced more negative events (P<0·0001) and perceived them with higher ratings (P<0·0001). Each group had similar coping ability In terms of the number of coping methods and magnitude of utilization of these methods. Similarly, Graves' disease patients reported more dally hassle (P<0·0001) and had higher hassle scores (P<0·0001). CONCLUSION Patients with Graves' disease experienced greater psychological stress and adverse events prior to the onset of the disease. As stress may alter the immune system, it could play an important role in precipitating the disease in subjects predisposed to auto-immune thyroid disorders.     

Regenerative peripheral nerve interfaces for real-time,proportional control of a Neuroprosthetic hand

Introduction

Regenerative peripheral nerve interfaces (RPNIs) are biological constructs which amplify neural signals and have shown long-term stability in rat models. Real-time control of a neuroprosthesis in rat models has not yet been demonstrated. The purpose of this study was to: a) design and validate a system for translating electromyography (EMG) signals from an RPNI in a rat model into real-time control of a neuroprosthetic hand, and; b) use the system to demonstrate RPNI proportional neuroprosthesis control.

Methods

Animals were randomly assigned to three experimental groups: (1) Control; (2) Denervated, and; (3) RPNI. In the RPNI group, the extensor digitorum longus (EDL) muscle was dissected free, denervated, transferred to the lateral thigh and neurotized with the residual end of the transected common peroneal nerve. Rats received tactile stimuli to the hind-limb via monofilaments, and electrodes were used to record EMG. Signals were filtered, rectified and integrated using a moving sample window. Processed EMG signals (iEMG) from RPNIs were validated against Control and Denervated group outputs.

Results

Voluntary reflexive rat movements produced signaling that activated the prosthesis in both the Control and RPNI groups, but produced no activation in the Denervated group. Signal-to-Noise ratio between hind-limb movement and resting iEMG was 3.55 for Controls and 3.81 for RPNIs. Both Control and RPNI groups exhibited a logarithmic iEMG increase with increased monofilament pressure, allowing graded prosthetic hand speed control (R²?=?0.758 and R²?=?0.802, respectively).

Conclusion

EMG signals were successfully acquired from RPNIs and translated into real-time neuroprosthetic control. Signal contamination from muscles adjacent to the RPNI was minimal. RPNI constructs provided reliable proportional prosthetic hand control.