Biomedical subjects

F Hayashi

Publications and source records attributed to F Hayashi.

At least 37 records · Page 2Linked to original sources

PubMed2000

Phosphorylation by cyclin-dependent protein kinase 5 of the regulatory subunit of retinal cGMP phosphodiesterase. I. Identification of the kinase and its role in the turnoff of phosphodiesterase in vitro.

Cyclic GMP phosphodiesterase (PDE) is an essential component in retinal phototransduction. PDE is regulated by Pgamma, the regulatory subunit of PDE, and GTP/Talpha, the GTP-bound alpha subunit of transducin. In previous studies (Tsuboi, S., Matsumoto, H. , Jackson, K. W., Tsujimoto, K., Williamas, T., and Yamazaki, A. (1994) J. Biol. Chem. 269, 15016-15023; Tsuboi, S., Matsumoto, H., and Yamazaki, A. (1994) J. Biol. Chem. 269, 15024-15029), we showed that Pgamma is phosphorylated by a previously unknown kinase (Pgamma kinase) in a GTP-dependent manner in photoreceptor outer segment membranes. We also showed that phosphorylated Pgamma loses its ability to interact with GTP/Talpha, but gains a 10-15 times higher ability to inhibit GTP/Talpha-activated PDE than that of nonphosphorylated Pgamma. Thus, we propose that the Pgamma phosphorylation is probably involved in the recovery phase of phototransduction through shut off of GTP/Talpha-activated PDE. Here we demonstrate that all known Pgammas preserve a consensus motif for cyclin-dependent protein kinase 5 (Cdk5), a protein kinase believed to be involved in neuronal cell development, and that Pgamma kinase is Cdk5 complexed with p35, a neuronal Cdk5 activator. Mutational analysis of Pgamma indicates that all known Pgammas contain a P-X-T-P-R sequence and that this sequence is required for the Pgamma phosphorylation by Pgamma kinase. In three different column chromatographies of a cytosolic fraction of frog photoreceptor outer segments, the Pgamma kinase activity exactly coelutes with Cdk5 and p35. The Pgamma kinase activity ( approximately 85%) is also immunoprecipitated by a Cdk5-specific antibody, and the immunoprecipitate phosphorylates Pgamma. Finally, recombinant Cdk5/p35, which were expressed using clones from a bovine retina cDNA library, phosphorylates Pgamma in frog outer segment membranes in a GTP-dependent manner. These observations suggest that Cdk5 is probably involved in the recovery phase of phototransduction through phosphorylation of Pgamma complexed with GTP/Talpha in mature vertebrate retinal photoreceptors.

3',5'-Cyclic-GMP Phosphodiesterases↗

PubMed2000

Phosphorylation by cyclin-dependent protein kinase 5 of the regulatory subunit of retinal cGMP phosphodiesterase. II. Its role in the turnoff of phosphodiesterase in vivo.

Retinal cGMP phosphodiesterase (PDE) is regulated by Pgamma, the regulatory subunit of PDE, and GTP/Talpha, the GTP-bound alpha subunit of transducin. In the accompanying paper (Matsuura, I., Bondarenko, V. A., Maeda, T., Kachi, S., Yamazaki, M., Usukura, J., Hayashi, F., and Yamazaki, A. (2000) J. Biol. Chem. 275, 32950-32957), we have shown that all known Pgammas contain a specific phosphorylation motif for cyclin-dependent protein kinase 5 (Cdk5) and that the unknown kinase is Cdk5 complexed with its activator. Here, using frog rod photoreceptor outer segments (ROS) isolated by a new method, we show that Cdk5 is involved in light-dependent Pgamma phosphorylation in vivo. Under dark conditions only negligible amounts of Pgamma were phosphorylated. However, under illumination that bleached less than 0.3% of the rhodopsin, approximately 4% of the total Pgamma was phosphorylated in less than 10 s. Pgamma dephosphorylation occurred in less than 1 s after the light was turned off. Analysis of the phosphorylated amino acid, inhibition of Pgamma phosphorylation by Cdk inhibitors in vivo and in vitro, and two-dimensional peptide map analysis of Pgamma phosphorylated in vivo and in vitro indicate that Cdk5 phosphorylates a Pgamma threonine in the same manner in vivo and in vitro. These observations, together with immunological data showing the presence of Cdk5 in ROS, suggest that Cdk5 is involved in light-dependent Pgamma phosphorylation in ROS and that the phosphorylation is significant and reversible. In an homogenate of frog ROS, PDE activated by light/guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS) was inhibited by Pgamma alone, but not by Pgamma complexed with GDP/Talpha or GTPgammaS/Talpha. Under these conditions, Pgamma phosphorylated by Cdk5 inhibited the light/GTPgammaS-activated PDE even in the presence of GTPgammaS/Talpha. These observations suggest that phosphorylated Pgamma interacts with and inhibits light/GTPgammaS-activated PDE, but does not interact with GTPgammaS/Talpha in the homogenate. Together, our results strongly suggest that after activation of PDE by light/GTP, Pgamma is phosphorylated by Cdk5 and the phosphorylated Pgamma inhibits GTP/Talpha-activated PDE, even in the presence of GTP/Talpha in ROS.

3',5'-Cyclic-GMP Phosphodiesterases↗

PubMed2000

Serine 6 of Lck tyrosine kinase: a critical site for Lck myristoylation, membrane localization, and function in T lymphocytes.

Lck is a member of the Src family kinases expressed predominantly in T cells, and plays a pivotal role in TCR-mediated signal transduction. Myristoylation of glysine 2 in the N-terminal Src homology 4 (SH4) domain of Lck is essential for membrane localization and function. In this study, we examined a site within the SH4 domain of Lck regulating myristoylation, membrane localization, and function of Lck. A Lck mutant in which serine 6 (Ser6) was substituted by an alanine was almost completely cytosolic in COS-7 cells, and this change of localization was associated with a drastic inhibition of myristoylation in this mutant. To assess the role of Ser6 of Lck in T cell function, we established stable transfectants expressing various Lck mutants using Lck-negative JCaM1 cells. The Lck mutant of Ser6 to alanine, most of which did not target to the plasma membrane, was not able to reconstitute TCR-mediated signaling events in JCaM1 cells, as analyzed by tyrosine phosphorylation of intracellular proteins and CD69 expression. These results demonstrate that Ser6 is a critical factor for Lck myristoylation, membrane localization, and function in T cells, presumably because the residue is important for N-myristoyl transferase recognition.

Alanine↗

PubMed2000

Influence of astigmatism on multifocal and monofocal intraocular lenses.

PURPOSE: To examine the influence of astigmatism on the visual acuity of patients with multifocal and monofocal intraocular lenses. METHODS: Thirty eyes of 30 patients who underwent five-zone refractive multifocal intraocular lens implantation and 30 eyes of 30 age-matched patients who had monofocal intraocular lens implantation were included. The visual acuities of these patients at 5.0, 3.0, 2.0, 1.0, 0.7, 0.5, and 0.3 m were measured by means of an all-distance vision tester after addition of a cylindrical lens of 0, 0.5, 1.0, 1.5, 2.0, and 2.5 diopters. RESULTS: Mean visual acuity at all distances decreased in proportion to the diopters of astigmatism in both the multifocal and monofocal intraocular lens groups. When astigmatism was 0.5, 1.0, or 1.5 diopters, distance visual acuity in the multifocal group was significantly worse than that in the monofocal group; with astigmatism of 2.0 diopters or more, intermediate visual acuity was also worse in the multifocal group. In contrast, near visual acuity in the multifocal group was significantly better than that in the monofocal group at all astigmatic diopters. When astigmatism was within 1.0 diopter, visual acuity in the multifocal group reached 20/29 at distance and 20/50 at near. Mean contrast sensitivity was also worse in the multifocal group than in the monofocal group. CONCLUSIONS: Both distance and intermediate visual acuity deterioration caused by astigmatism was greater with a multifocal intraocular lens than with a monofocal intraocular lens, whereas near visual acuity was better with the multifocal intraocular lens. When astigmatism was within 1.0 diopter, eyes with a multifocal intraocular lens achieved good visual acuity at both distance and near.

Accommodation, Ocular↗

PubMed2000

Changes in anterior chamber angle width and depth after intraocular lens implantation in eyes with glaucoma.

OBJECTIVE: To examine the changes in anterior chamber angle width and depth induced by intraocular lens (IOL) implantation in eyes with angle-closure glaucoma (ACG), in eyes with open-angle glaucoma (OAG), and in eyes with no evidence of glaucoma or ocular hypertension. DESIGN: A comparative, nonrandomized, interventional study. PARTICIPANTS: Seventy-seven eyes with ACG, 73 eyes with OAG, and 74 control eyes undergoing cataract extraction and IOL implantation. INTERVENTION: All eyes underwent phacoemulsification and soft acrylic IOL implantation. MAIN OUTCOME MEASURES: The angle width and depth of the anterior chamber were measured using a Scheimpflug videophotography system before surgery, and at 1 week and at 1, 3, 6, 9, and 12 months after surgery. RESULTS: Before surgery, the mean anterior chamber angle width and depth in the ACG group was less than that in either the OAG or control groups by approximately 10 degrees in angle width and 1.0 mm in depth (P < 0.0001). After cataract extraction and IOL implantation, the angle width and depth increased significantly in all three groups (P < 0.0001). Although the width and depth in the ACG group were still smaller than that in the other groups, the differences decreased to 2 degrees for angle width and 0.3 mm for depth. In addition, no significant differences were found in these values between the OAG and control groups before or after surgery. Furthermore, no significant changes were observed in the angle width or depth in any of the three groups throughout the postoperative observation period. As expected, the mean preoperative intraocular pressure (IOP) in the ACG and OAG groups was higher than that in the control group. After cataract surgery, however, the mean IOP decreased significantly and was almost the same in all three groups at 1, 6, and 12 months after surgery. CONCLUSIONS: The width and depth of the anterior chamber angle in eyes with ACG increased significantly after cataract extraction and IOL implantation and became similar to that in eyes with OAG and that in normal eyes, which may lead to the decrease in IOP seen in the postoperative period. No significant changes were observed in angle width and depth in any of the three groups after surgery.

Aged↗

PubMed2000

Inhibitory mechanisms in hypoxic respiratory depression studied in an in vitro preparation.

A medullary-spinal cord preparation without the pons isolated from the neonatal rat was used to investigate the role of inhibitory neurotransmitters in the respiratory depression induced by hypoxia (hypoxic respiratory depression; HRD). The burst frequency (C(4)-f) and peak amplitudes of the integrated activity of the C(4) roots (integral C(4)) and of the hypoglossal nerve (integral XII) were recorded. A marked decrease in C(4)-f (to 36+/-6% of control, P<0. 05) with no change in the peak amplitudes of integral C(4) or integral XII was observed 17-21 min after superfusion with hypoxic CSF bubbled with 5% CO(2) in N(2). Antagonists of GABA(A) (bicuculline; 10 microM), GABA(B) (phaclofen; 0.2-0.5 mM), glycine (strychnine; 10 mM), adenosine (aminophylline; 100 mM) or opioid (naloxone; 1 mM) receptors were added to the bathing solution to block inhibitory synaptic transmission. Among these antagonists, only strychnine and naloxone alleviated HRD reducing the decline in C(4)-f to 57+/-11 and 53+/-6%, respectively (P<0.05). Posthypoxic neural arrest (PHNA) following resumption of oxygenation was shortened by the application of aminophylline, strychnine or naloxone (by 91+/-17, 96+/-25 and 40+/-6 s, respectively, P<0.05). These findings indicate that the reduction in the frequency component of HRD depends on glycinergic and opioid-mediated neuronal inhibition in an in vitro medullary spinal cord preparation. It was also observed that the duration of PHNA was positively correlated with the severity of the fall in C(4)-f (r=0.60, P<0.01).

Animals↗

PubMed2000

Fourier analysis of irregular astigmatism after implantation of 3 types of intraocular lenses.

PURPOSE: To evaluate irregular astigmatism after silicone, acrylic, and poly(methyl methacrylate) (PMMA) intraocular lens (IOL) implantation using Fourier analysis of videokeratography data. METHODS: Two hundred forty eyes having phacoemulsification and IOL implantation were randomly assigned to 1 of 3 groups: 3.5 mm incision and silicone IOL, 4.1 mm incision and acrylic IOL, or 6.5 mm incision and PMMA IOL. All eyes had videokeratographic examinations preoperatively and 2, 4, and 10 days and 1 and 3 months postoperatively. The dioptric data of the central cornea were decomposed into spherical equivalent, regular astigmatism, and irregular astigmatism (decentration and higher-order irregularity) components using Fourier analysis. RESULTS: Regular astigmatism in the PMMA group was greater than in the silicone and acrylic groups. Decentration in all 3 groups increased significantly postoperatively but virtually returned to preoperative levels by 10 days. No significant difference was observed among the 3 groups. Higher-order irregularity significantly increased after surgery in all 3 groups. The higher-order irregularity in the PMMA group persisted for up to 1 month, while that in the silicone and acrylic groups returned to preoperative levels by 4 days, resulting in significant differences between these groups 10 days and 1 month after IOL implantation. CONCLUSION: Irregular astigmatism, both the decentration and higher-order irregularity components, increased significantly after 3 types of scleral tunnel incisions for silicone, acrylic, or PMMA IOL implantation but returned to preoperative levels soon after surgery except for the higher order irregularity after PMMA IOL implantation.

Acrylic Resins↗

PubMed2000

Electric double-sleeved vacuuming microtrephine for lens refilling.

An automated electric double-sleeved vacuuming microtrephine has been developed that consistently creates a 0.8 to 1.0 mm tear-free hole in the anterior lens capsule. This hole is required for lens refilling to retain accommodation after cataract removal. The trephine is double-sleeved. The stainless-steel outer sleeve vacuums the internal anterior capsule; the stainless steel rotating inner sleeve, which has a blade on the head, then slides forward and cuts the taut anterior capsule. All manipulations are controlled automatically by depressing a foot pedal. Using this instrument, a 0. 8 to 1.0 mm tear-free microhole in the anterior capsule was created successfully in 80% of excised pig (n = 30) and eye-bank (n = 10) eyes. This trephine will be useful for lens refilling in the future.

Animals↗

PubMed2000

Detection of Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis in dental plaque samples from children 2 to 12 years of age.

The purpose of this study was to detect the presence of Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis in plaque samples from 104 children, collected from their toothbrushes using a polymerase chain reaction (PCR). The age range of all subjects was 2-12 years. 21, 73 and 10 children with healthy gingiva, gingivitis and periodontitis respectively were selected. Plaque samples were collected from all erupted teeth sites using a sterile toothbrush. The mean concentration of DNA recovered from brushing plaque samples was approximately 660 microg/ml, which was sufficient for performing a PCR-based survey. Both A. actinomycetemcomitans and P. gingivalis were detected in the primary and mixed dentition. The prevalence of A. actinomycetemcomitans in healthy subjects was 4.8%, and those with gingivitis and periodontitis was 6.8% and 20.0% respectively, while the prevalence of P. gingivalis was 4.8% in healthy subjects, and 9.6% and 20.0% in those with gingivitis and periodontitis, respectively. Our survey, using a toothbrush, indicated that A. actinomycetemcomitans and P. gingivalis are rarely present in oral cavities of healthy children.

Aggregatibacter actinomycetemcomitans↗

PubMed2000

Structural change of bovine retinal cGMP phosphodiesterase by release of its gamma subunit: direct imaging by improved low angle rotary shadowing.

Cyclic GMP phosphodiesterase (PDE), a key enzyme for phototransduction, contains two catalytic subunits, Palpha and Pbeta, and two identical regulatory subunits, Pgammas. Neither the structure of the subunits of PDE nor their changes in structure during PDE regulation have been known. Here, improved low angle rotary shadowing was applied to depict the three-dimensional structure of bovine PDE (Palphabetagammagamma) and its changes by Pgamma release. Palphabetagammagamma and Palphabetagamma were isolated from photoreceptor membranes after treatment with a hydrolysis-resistant GTP analogue, and Palphabeta was prepared from Palphabetagammagamma tryptic digestion. Images of Palphabetagammagamma consisted of two crooked strands. These two strands faced each other to make a ring shape, but this ring structure was bent at the centre line between the two strands. In Palphabetagamma, one of these strands changed its shape toward reducing the central space of the ring structure. This ring appeared to be more bent at the centre line. In Palphabeta, both strands changed their shape such that the ring structure appeared to be a twisted quasi ring shape. These observations suggest that in Palphabetagammagamma each Pgamma is complexed with a catalytic subunit, and that the shapes of Palpha and Pbeta are drastically changed by the Pgamma release. These shape changes are no doubt crucial for various PDE regulations, such as activation of cGMP hydrolysis by Palphabeta, interaction of Palphabeta with GARP2 and a GARP2-like protein and cGMP binding to non-catalytic sites on Palphabeta.

3',5'-Cyclic-GMP Phosphodiesterases↗

PubMed2000

Oscillation and regulation of proline content by P5CS and ProDH gene expressions in the light/dark cycles in Arabidopsis thaliana L.

The fluctuation of proline content, and protein and mRNA levels of delta1-pyrroline-5-carboxylate synthetase (P5CS) and proline dehydrogenase (ProDH), both of which are involved in proline biosynthesis and degradation, in the shoots of Arabidopsis grown in light/dark cycles were demonstrated under salt-stressed and unstressed conditions. Proline content, as well as proteins and mRNAs of these enzymes, clearly oscillated in the light/dark cycles under the stressed and unstressed conditions. A reciprocal relationship between P5CS and ProDH was observed. Protein levels of P5CS and ProDH were well synchronized with their mRNA levels, although the fluctuation of protein levels was not as significant as that of their mRNA levels. Both mRNA and protein levels of the two enzymes as well as the proline content did not oscillate under the continuous light or the dark conditions. Thus, P5CS and ProDH gene expressions seemed to be involved in light irradiation. Moreover, relative water content (RWC) in the plants oscillated in the light/dark cycles. The fluctuations of proline content in shoot reversely responded to that of RWC. It is suggested that the expression of two genes responds sensitively to a subtle change of cellular water status, and accumulated proline keeps the osmotic balance between cells and the outer environment.

Arabidopsis↗

PubMed2000

Responses of tracheobronchial receptors to inhaled furosemide in anesthetized rats.

Inhalation of furosemide, a loop diuretic, has shown favorable effects on experimentally induced cough, bronchoconstriction, and dyspnea. The effect of inhaled furosemide on tracheobronchial receptors was studied in anesthetized, spontaneously breathing rats. Single unit or pauci unit activity was recorded from the right vagus nerve. Tracheobronchial receptors were classified into slowly and rapidly adapting receptors (SARs and RARs, respectively), based on their adaptation index (AI), which was derived from the decrease in spike frequency (sf) over 2 s, expressed as a percentage of the peak firing rate. There were 43 SARs (AI </= 25%) and eight RARs (AI >/= 50%). Inhalation of furosemide (n = 29) increased the slope of airway pressure (Paw) versus sf of SARs from 8.6 to 14.8 Hz/cm H(2)O with an increase in sf at Paw = 0 cm H(2)O from 18.0 to 49.5 Hz, resulting in an upward shift of the line. Neither inhalation of vehicle (n = 9) nor intravenous injection of furosemide (n = 5) changed this relationship. In addition, inhalation of furosemide attenuated the activity of RARs. These findings indicate that SARs are sensitized and RARs desensitized by inhalation of furosemide. We discuss possible mechanisms for this, and its relevance to clinical problems of dyspnea, bronchoconstriction, and cough.

Administration, Inhalation↗

PubMed2000

Circulating adrenomedullin in erythrocopietin-induced hypertension.

Levels of adrenomedullin (AM) have been shown to be elevated in hypertension and chronic renal failure, suggesting that AM plays a role in the pathogenesis of these diseases. The objective of the present study was to investigate whether circulating AM is involved in erythropoietin (Epo)-induced hypertension in patients with renal anemia due to progressive renal disease. Following treatment with 6,000 IU of Epo once a week, the hematocrit (Ht) rose significantly from 25.9+/-4.0 to 33.4+/-3.3% (n=54, p<0.001) with an overall rate of increase in Ht of 0.43+/-0.04%/week. In response to treatment with Epo, a rise in mean blood pressure of >10 mmHg (Epo-induced hypertension) was found in 22% (12/54 cases) of the patients enrolled. There was no difference in the rate of Ht increase between patients with and without Epo-induced hypertension. There was a significant positive correlation between mature AM and serum creatinine (Cr) concentration before treatment with Epo. However, no correlation was found between the plasma concentration of total AM and serum Cr concentration. Long-term treatment with Epo did not influence plasma concentration of either mature AM or total AM in patients developing hypertension during the study period. These results suggest that circulating AM may play a role in the progression of renal disease. However, the present study does not support the notion that circulating AM is associated with the pathogenesis of Epo-induced hypertension. It is too early yet to claim that there is no AM-mediated mechanism in Epo-induced hypertension.

Adrenomedullin↗

PubMed2000

Neuronal mechanisms mediating the integration of respiratory responses to hypoxia.

The activation of peripheral chemoreceptors by hypoxia or electrical stimulation of the carotid sinus nerve elicited a hypoxic respiratory response consisting of both stimulatory and subsequent or simultaneous inhibitory components (hypoxic respiratory stimulation and depression). Both components have different time domains of responses (time-dependent response), providing an integrated respiratory response to hypoxia. This review has focused on the neuroanatomical and neurophysiological correlations responsible for these responses and their neuropharmacological mechanisms. Hypoxic respiratory depression is characterized by the initial activation of respiration followed by a progressive and gradual decline in ventilation during prolonged and/or severe hypoxic exposure (biphasic response). The responsible mechanisms for the depression are located within the central nervous system and may be dependent upon activity from peripheral chemoreceptor. Two underlying mechanisms contributing to the depression have been advocated. (1) Change in synaptic transmission: Within the neuronal network controlling the hypoxic respiratory response, hypoxia might induce the enhancement of inhibitory neurotransmission (modulation), disfacilitation of excitatory neruotransmission or both. (2) Change in the membrane property of respiratory neurons: Hypoxia might suppress the membrane excitability of respiratory neurons composing the hypoxic respiratory response via modulating ion channels, leading to hyperpolarization or depolarization blocking of the neurons. However, the quantitative aspects of Pao(2) (degree and duration of hypoxic exposure) to induce these changes and the susceptibility of both mechanisms to the Pao(2) level have not yet been clearly elucidated.

Animals↗

PubMed1999

Suppression of GTP/T alpha-dependent activation of cGMP phosphodiesterase by ADP-ribosylation by its gamma subunit in amphibian rod photoreceptor membranes.

Our previous study has shown that P gamma, the regulatory subunit of cGMP phosphodiesterase (PDE), is ADP-ribosylated by endogenous ADP-ribosyltransferase when P gamma is free or complexed with the catalytic subunits of PDE in amphibian rod photoreceptor membranes. The P gamma domain containing ADP-ribosylated arginines was shown to be involved in its interaction with T alpha, a key interaction for PDE activation. In this study, we describe a possible function of the P gamma ADP-ribosylation in the GTP/T alpha-dependent PDE activation. When rod membranes were preincubated with or without NAD and washed with a buffer containing GTP, the PDE activity of NAD-preincubated membranes was increased by the GTP-washing only to approximately 50% of that of membranes preincubated without NAD. The P gamma release by the GTP-washing from these NAD-preincubated membranes was also suppressed to approximately 50% of that preincubated without NAD. Taking into consideration that approximately 50% of P gamma is ADP-ribosylated under these conditions, these observations suggest that the ADP-ribosylated P gamma cannot interact with GTP/T alpha. We have also shown that a soluble fraction of ROS contains an enzyme(s) to release the radioactivity of [32P]ADP-ribosylated P gamma in concentration- and time-dependent manners, suggesting that the P gamma ADP-ribosylation is reversible. Rod ADP-ribosyltransferase solubilized from membranes by phosphatidylinositol-specific phospholipase C was separated into two fractions by ion-exchange columns. Biochemical characterization of these two fractions, including measurement of the Km for NAD and P gamma, estimation of their molecular masses, ADP-ribosylation of P gamma arginine mutants, effects of ADP-ribosyltransferase inhibitors on the P gamma ADP-ribosylation, and effects of salts and pH on the P gamma ADP-ribosylation, indicates that rod ADP-ribosyltransferase contains two isozymes, and that these two isozymes have similar properties for the P gamma ADP-ribosylation. Our observations strongly suggest that the negative regulation of PDE through the reversible P gamma ADP-ribosylation may function in the phototransduction mechanism.

3',5'-Cyclic-GMP Phosphodiesterases↗

PubMed1999

Activation of retinal guanylyl cyclase-1 by Ca2+-binding proteins involves its dimerization.

Retinal guanylyl cyclase-1 (retGC-1), a key enzyme in phototransduction, is activated by guanylyl cyclase-activating proteins (GCAPs) if [Ca2+] is less than 300 nM. The activation is believed to be essential for the recovery of photoreceptors to the dark state; however, the molecular mechanism of the activation is unknown. Here, we report that dimerization of retGC-1 is involved in its activation by GCAPs. The GC activity and the formation of a 210-kDa cross-linked product of retGC-1 were monitored in bovine rod outer segment homogenates, GCAPs-free bovine rod outer segment membranes and recombinant bovine retGC-1 expressed in COS-7 cells. In addition to recombinant bovine GCAPs, constitutively active mutants of GCAPs that activate retGC-1 in a [Ca2+]-independent manner and bovine brain S100b that activates retGC-1 in the presence of approximately 10 microM [Ca2+] were used to investigate whether these activations take place through a similar mechanism, and whether [Ca2+] is directly involved in the dimerization. We found that a monomeric form of retGC-1 ( approximately 110 kDa) was mainly observed whenever GC activity was at basal or low levels. However, the 210-kDa product was increased whenever the GC activity was stimulated by any Ca2+-binding proteins used. We also found that [Ca2+] did not directly regulate the formation of the 210-kDa product. The 210-kDa product was detected in a purified GC preparation and did not contain GCAPs even when the formation of the 210-kDa product was stimulated by GCAPs. These data strongly suggest that the 210-kDa cross-linked product is a homodimer of retGC-1. We conclude that inactive retGC-1 is predominantly a monomeric form, and that dimerization of retGC-1 may be an essential step for its activation by active forms of GCAPs.

Animals↗

PubMed1999

Multifunctional laryngeal motoneurons: an intracellular study in the cat.

We studied the patterns of membrane potential changes in laryngeal motoneurons (LMs) during vocalization, coughing, swallowing, sneezing, and the aspiration reflex in decerebrate paralyzed cats. LMs, identified by antidromic activation from the recurrent laryngeal nerve, were expiratory (ELMs) or inspiratory (ILMs) cells that depolarized during their respective phases in eupnea. During vocalization, most ELMs depolarized and most ILMs hyperpolarized. Some ILMs depolarized slightly during vocalization. During coughing, ELMs depolarized abruptly at the transition from the inspiratory to the expiratory phase. In one-third of ELMs, this depolarization persisted throughout the abdominal burst. In the remainder ("type A"), it was interrupted by a transient repolarization. ILMs exhibited a membrane potential trajectory opposite to that of type A ELMs during coughing. During swallowing, the membrane potential of ELMs decreased transiently at the onset of the hypoglossal burst and then depolarized strongly during the burst. ILMs hyperpolarized sharply at the onset of the burst and depolarized as hypoglossal activity ceased. During sneezing, ELMs and ILMs exhibited membrane potential changes similar to those of type A ELMs and ILMs during coughing. During the aspiration reflex, ELMs and ILMs exhibited bell-shaped hyperpolarization and depolarization trajectories, respectively. We conclude that central drives to LMs, consisting of complex combinations of excitation and inhibition, vary during vocalization and upper airway defensive reflexes. This study provides data for analysis of the neuronal networks that produce these various behaviors and analysis of network reorganization caused by changes in dynamic connections between the respiratory and nonrespiratory neuronal networks.

Animals↗

PubMed1999

Optimal distances for squirrels to transport and hoard walnuts.

Walnuts, Juglans airanthifolia, are an important food item for Japanese squirrels, Sciurus lis, which hoard them for later use. We set up feeders in small walnut patches in secondary forest, mounted radiotransmitters on nuts, placed them on the feeders and tracked them when they were cached. Of 720 nuts placed on the feeders over a 3-year period, 32.6% were eaten immediately and 50.6% were scatter-hoarded 0-168 m from the feeders. With increasing distance to the cache site, the squirrels took longer to return to the feeders (range 0.35-35 min). However, the further away a cache was, the less likely it was to be stolen by conspecifics or other nut-eating animals: nuts we hoarded ourselves disappeared more quickly from near the source trees. Squirrels took nuts further away later in the season when caches were lost at a higher rate. Using a simple model maximizing the rate of energy gain per unit time taken to search for and hoard the nuts at different distances from the source, we discuss whether squirrels alter the transport distance of hoarding nuts according to cache survival rate and/or nut availability. Copyright 1999 The Association for the Study of Animal Behaviour.

Journal Article↗