A Component of Retinal Light Adaptation Mediated by the Thyroid Hormone Cascade

Analysis with DNA-microrrays and real time PCR show that several genes involved in the thyroid hormone cascade, such as deiodinase 2 and 3 (Dio2 and Dio3) are differentially regulated by the circadian clock and by changes of the ambient light. The expression level of Dio2 in adult rats (2–3 months of age) kept continuously in darkness is modulated by the circadian clock and is up-regulated by 2 fold at midday. When the diurnal ambient light was on, the expression level of Dio2 increased by 4–8 fold and a consequent increase of the related protein was detected around the nuclei of retinal photoreceptors and of neurons in inner and outer nuclear layers. The expression level of Dio3 had a different temporal pattern and was down-regulated by diurnal light. Our results suggest that DIO2 and DIO3 have a role not only in the developing retina but also in the adult retina and are powerfully regulated by light. As the thyroid hormone is a ligand-inducible transcription factor controlling the expression of several target genes, the transcriptional activation of Dio2 could be a novel genomic component of light adaptation

Structure and Function of the Hair Cell Ribbon Synapse

Faithful information transfer at the hair cell afferent synapse requires synaptic transmission to be both reliable and temporally precise. The release of neurotransmitter must exhibit both rapid on and off kinetics to accurately follow acoustic stimuli with a periodicity of 1 ms or less. To ensure such remarkable temporal fidelity, the cochlear hair cell afferent synapse undoubtedly relies on unique cellular and molecular specializations. While the electron microscopy hallmark of the hair cell afferent synapse — the electron-dense synaptic ribbon or synaptic body — has been recognized for decades, dissection of the synapse’s molecular make-up has only just begun. Recent cell physiology studies have added important insights into the synaptic mechanisms underlying fidelity and reliability of sound coding. The presence of the synaptic ribbon links afferent synapses of cochlear and vestibular hair cells to photoreceptors and bipolar neurons of the retina. This review focuses on major advances in understanding the hair cell afferent synapse molecular anatomy and function that have been achieved during the past years

Cyclic AMP diffusion coefficient in frog olfactory cilia.

Cyclic AMP (cAMP) is one of the intracellular messengers that mediate odorant signal transduction in vertebrate olfactory cilia. Therefore, the diffusion coefficient of cAMP in olfactory cilia is an important factor in the transduction of the odorous signal. We have employed the excised cilium preparation from the grass frog (Rana pipiens) to measure the cAMP diffusion coefficient. In this preparation an olfactory cilium is drawn into a patch pipette and a gigaseal is formed at the base of the cilium. Subsequently the cilium is excised, allowing bath cAMP to diffuse into the cilium and activate the cyclic nucleotide-gated channels on the plasma membrane. In order to estimate the cAMP diffusion coefficient, we analyzed the kinetics of the currents elicited by step changes in the bath cAMP concentration in the absence of cAMP hydrolysis. Under such conditions, the kinetics of the cAMP-activated currents has a simple dependence on the diffusion coefficient. From the analysis we have obtained a cAMP diffusion coefficient of 2.7 +/- 0.2. 10(-6) cm2 s-1 for frog olfactory cilia. This value is similar to the expected value in aqueous solution, suggesting that there are no significant diffusional barriers inside olfactory cilia. At cAMP concentrations higher than 5 microM, diffusion slowed considerably, suggesting the presence of buffering by immobile cAMP binding sites. A plausible physiological function of such buffering sites would be to prolong the response of the cell to strong stimuli

Cyclic GMP diffusion coefficient in rod photoreceptor outer segments.

Cyclic GMP (cGMP) is the intracellular messenger that mediates phototransduction in retinal rods. As photoisomerizations of rhodopsin molecules are local events, the longitudinal diffusion of cGMP in the rod outer segment should be a contributing factor to the response of the cell to light. We have employed the truncated rod outer segment preparation from bullfrog (Rana catesbeiana) and tiger salamander (Ambystoma tigrinum) to measure the cGMP diffusion coefficient. In this preparation, the distal portion of a rod outer segment was drawn into a suction pipette for measuring membrane current, and the rest of the cell was then sheared off with a glass probe, allowing bath cGMP to diffuse into the outer segment and activate the cGMP-gated channels on the surface membrane. Addition and removal of bath cGMP were fast enough to produce effectively step changes in cGMP concentration at the open end of the outer segment. When cGMP hydrolysis is inhibited by isobutylmethylxanthine (IBMX), the equation for the diffusion of cGMP inside the truncated rod outer segment has a simple analytical solution, which we have used to analyze the rise and decay kinetics of the cGMP-elicited currents. From these measurements we have obtained a cGMP diffusion coefficient of approximately 70 x 10(-8) cm2 s-1 for bullfrog rods and approximately 60 x 10(-8) cm2 s-1 for tiger salamander rods. These values are six to seven times lower than the expected value in aqueous solution. The estimated diffusion coefficient is the same at high (20-1000 microM) and low (5-10 microM) concentrations of cGMP, suggesting no significant effect from buffering over these concentration ranges

Diffusion coefficient of the cyclic GMP analog 8-(fluoresceinyl)thioguanosine 3',5' cyclic monophosphate in the salamander rod outer segment.

Cyclic GMP (cGMP) is the intracellular messenger mediating phototransduction in retinal rods, with its longitudinal diffusion in the rod outer segment (ROS) likely to be a factor in determining light sensitivity. From the kinetics of cGMP-activated currents in the truncated ROS of the salamander (Ambystoma tigrinum), the cGMP diffusion coefficient was previously estimated to be approximately 60 x 10(-8) cm2 s-1. On the other hand, fluorescence measurements in intact salamander ROS using 8-(fluoresceinyl)thioguanosine 3',5'-cyclic monophosphate (Fl-cGMP) led to a diffusion coefficient for this compound of 1 x 10(-8) cm2 s-1; after corrections for differences in size and in binding to cellular components between cGMP and Fl-cGMP, this gave an upper limit of 11 x 10(-8) cm2 s-1 for the cGMP diffusion coefficient. To properly compare the two sets of measurements, we have examined the diffusion of Fl-cGMP in the truncated ROS. From the kinetics of Fl-cGMP-activated currents, we have obtained a diffusion coefficient of 3 x 10(-8) cm2 s-1 for this analog; the cGMP diffusion coefficient measured from the same truncated ROSs was approximately 80 x 10(-8) cm2 s-1. Thus, a factor of 27 appears appropriate for correcting differences in size and intracellular binding between cGMP and Fl-cGMP. Application of this correction factor to the Fl-cGMP diffusion coefficient measurements by Olson and Pugh (1993) gives a cGMP diffusion coefficient of approximately 30 x 10(-8) cm2 s-1, in reasonable agreement with the value measured from the truncated ROS

Free magnesium concentration in salamander photoreceptor outer segments

Magnesium ions (Mg2+) play an important role in biochemical functions. In vertebrate photoreceptor outer segments, numerous reactions utilize MgGTP and MgATP, and Mg2+ also regulates several of the phototransduction enzymes. Although Mg2+ can pass through light-sensitive channels under certain conditions, no clear extrusion mechanism has been identified and removing extracellular Mg2+ has no significant effect on the light sensitivity or the kinetics of the photoresponse. We have used the fluorescent Mg2+ dye Furaptra to directly measure and monitor the free Mg2+ concentration in photoreceptor outer segments and examine whether the free Mg2+ concentration changes under physiological conditions. Resting free Mg2+ concentrations in bleached salamander rod and cone photoreceptor cell outer segments were 0.86 ± 0.06 and 0.81 ± 0.09 mm, respectively. The outer segment free Mg2+ concentration was not significantly affected by changes in extracellular pH, Ca2+ and Na+, excluding a significant role for the respective exchangers in the regulation of Mg2+ homeostasis. The resting free Mg2+ concentration was also not significantly affected by exposure to 0 Mg2+, suggesting the lack of significant basal Mg2+ flux. Opening the cGMP-gated channels led to a significant increase in the Mg2+ concentration in the absence of Na+ and Ca2+, but not in their presence, indicating that depolarization can cause a significant Mg2+ influx only in the absence of other permeant ions, but not under physiological conditions. Finally, light stimulation did not change the Mg2+ concentration in the outer segments of dark-adapted photoreceptors. The results suggest that there are no influx and efflux pathways that can significantly affect the Mg2+ concentration in the outer segment under physiological conditions. Therefore, it is unlikely that Mg2+ plays a significant role in the dynamic modulation of phototransduction

Satisfactory functional and radiological outcomes can be expected in young patients under 45 years old after open wedge high tibial osteotomy in a long-term follow-up

Purpose: To report the long-term outcomes of medial open wedge high tibial osteotomy (MOWHTO) for the treatment of medial compartment knee osteoarthritis in patients younger than 45 years old. It was hypothesized that the correction of knee alignment would result in preservation of knee function in a long-term follow-up. Methods: Patients under 45 years old, who underwent MOWHTO for symptomatic medial compartment knee osteoarthritis between 2001 and 2005 were retrospectively reviewed after a minimum of 10 years. The osteotomy was performed utilizing a locking plate without the use of bone graft. Patients were evaluated pre- and postoperatively using the International Knee Documentation Committee Score, the Oxford Knee Score, the Knee injury Osteoarthritis Outcome Score and the Short Form-12 Score. Standardized standing whole-limb radiographs were also obtained to assess mechanical tibiofemoral angle (mTFA) and the grade of osteoarthritis. Results: A total of 20 patients (18 males, 2 females, mean age 35.4 years) with a mean follow-up of 12.3 years were included in the study. During the follow-up period, one patient required conversion to total knee replacement (95% survival rate). All clinical outcome scores (IKDC, KOOS, OKS, and SF-12) significantly improved postoperatively (p < 0.05), with no significant deterioration over time. Preoperative varus alignment with an mTFA of − 5.8 ± 2.4° was corrected to 2.5 ± 1.9° immediately after surgery (p < 0.05), and remained 2.2 ± 1.7° at the last follow-up. Furthermore, no significant radiographic progression of osteoarthritis was observed. Conclusions: MWOHTO with a locking plate is an effective joint preservation method to treat medial compartment OA in active patients less than 45 years. Clinical and radiological results are satisfactory and the survival rate is 95%, 12 years after the procedure. Level of evidence: Level IV therapeutic, retrospective, cohort study. © 2017, European Society of Sports Traumatology, Knee Surgery, Arthroscopy (ESSKA)