Biomedical subjects

F Hayashi

Publications and source records attributed to F Hayashi.

At least 19 recordsLinked to original sources

PubMed2002

Short-term plasticity of descending synaptic input to phrenic motoneurons in rats.

Respiratory afferent stimulation can elicit increases in respiratory motor output that outlast the period of stimulation by seconds to minutes [short-term potentiation (STP)]. This study examined the potential contribution of spinal mechanisms to STP in anesthetized, vagotomized, paralyzed rats. After C(1) spinal cord transection, stimulus trains (100 Hz, 5-60 s) of the C(1)-C(2) lateral funiculus elicited STP of phrenic nerve activity that peaked several seconds poststimulation. Intracellular recording revealed that individual phrenic motoneurons exhibited one of three different responses to stimulation: 1) depolarization that peaked several seconds poststimulation, 2) depolarization during stimulation and then exponential repolarization after stimulation, and 3) bistable behavior in which motoneurons depolarized to a new, relatively stable level that was maintained after stimulus termination. During the STP, excitatory postsynaptic potentials elicited by single-stimulus pulses were larger and longer. In conclusion, repetitive activation of the descending inputs to phrenic motoneurons causes a short-lasting depolarization of phrenic motoneurons, and augmentation of excitatory postsynaptic potentials, consistent with a contribution to STP.

Animals↗

PubMed2001

The innate immune response to bacterial flagellin is mediated by Toll-like receptor 5.

The innate immune system recognizes pathogen-associated molecular patterns (PAMPs) that are expressed on infectious agents, but not on the host. Toll-like receptors (TLRs) recognize PAMPs and mediate the production of cytokines necessary for the development of effective immunity. Flagellin, a principal component of bacterial flagella, is a virulence factor that is recognized by the innate immune system in organisms as diverse as flies, plants and mammals. Here we report that mammalian TLR5 recognizes bacterial flagellin from both Gram-positive and Gram-negative bacteria, and that activation of the receptor mobilizes the nuclear factor NF-kappaB and stimulates tumour necrosis factor-alpha production. TLR5-stimulating activity was purified from Listeria monocytogenes culture supernatants and identified as flagellin by tandem mass spectrometry. Expression of L. monocytogenes flagellin in non-flagellated Escherichia coli conferred on the bacterium the ability to activate TLR5, whereas deletion of the flagellin genes from Salmonella typhimurium abrogated TLR5-stimulating activity. All known TLRs signal through the adaptor protein MyD88. Mice challenged with bacterial flagellin rapidly produced systemic interleukin-6, whereas MyD88-null mice did not respond to flagellin. Our data suggest that TLR5, a member of the evolutionarily conserved Toll-like receptor family, has evolved to permit mammals specifically to detect flagellated bacterial pathogens.

Adaptor Proteins, Signal Transducing↗

PubMed2001

Light- and guanosine 5'-3-O-(thio)triphosphate-sensitive localization of a G protein and its effector on detergent-resistant membrane rafts in rod photoreceptor outer segments.

Detergent-resistant membrane microdomains in the plasma membrane, known as lipid rafts, have been implicated in various cellular processes. We report here that a low-density Triton X-100-insoluble membrane (detergent-resistant membrane; DRM) fraction is present in bovine rod photoreceptor outer segments (ROS). In dark-adapted ROS, transducin and most of cGMP-phosphodiesterase (PDE) were detergent-soluble. When ROS membranes were exposed to light, however, a large portion of transducin localized in the DRM fraction. Furthermore, on addition of guanosine 5'-3-O-(thio)triphosphate (GTPgammaS) to light-bleached ROS, transducin became detergent-soluble again. PDE was not recruited to the DRM fraction after light stimulus alone, but simultaneous stimulation by light and GTPgammaS induced a massive translocation of all PDE subunits to the DRM. A cholesterol-removing reagent, methyl-beta-cyclodextrin, selectively but partially solubilized PDE from the DRM, suggesting that cholesterol contributes, at least in part, to the association of PDE with the DRM. By contrast, transducin was not extracted by the depletion of cholesterol. These data suggest that transducin and PDE are likely to perform their functions in phototransduction by changing their localization between two distinct lipid phases, rafts and surrounding fluid membrane, on disc membranes in an activation-dependent manner.

3',5'-Cyclic-GMP Phosphodiesterases↗

PubMed2001

Identification and characterization of a transcriptional regulator for the lck proximal promoter.

The lck gene encodes a protein-tyrosine kinase that plays a key role in signaling mediated through T cell receptor (TCR) and pre-TCR complexes. Transcription of the lck gene is regulated by two independent promoter elements: the proximal and distal promoters. Previous studies employing transgenic mice demonstrated that the sequence between -584 and -240 from the transcription start site in the mouse lck proximal promoter is required for its tissue-specific expression in the thymus. In this study, we demonstrate that a Krüppel-like zinc finger protein, mtbeta (BFCOL1, BERF-1, ZBP-89, ZNF148), previously cloned as a protein that binds to the CD3delta gene enhancer, binds to the -365 to -328 region of the lck proximal promoter. mtbeta is ubiquitously expressed in various cell lines and mouse tissues. Overexpressed mtbeta is more active in T-lineage cells than B-lineage cells for transactivating an artificial promoter consisting of the mtbeta binding site and a TATA box. Activity of the lck proximal promoter was significantly impaired by mutating the mtbeta binding site or by reducing mtbeta protein expression level by using antisense mRNA. Our results indicate that mtbeta activity is regulated in a tissue-specific manner and that mtbeta is a critical transactivator for the lck proximal promoter.

Animals↗

PubMed2001

A pivotal role of cysteine 3 of Lck tyrosine kinase for localization to glycolipid-enriched microdomains and T cell activation.

Lck, a Src family protein tyrosine kinase (PTKs), is post-translationally modified by palmitoylation, a process thought to regulate the biological function, membrane affinity and glycolipid-enriched microdomain (GEM) localization of this molecule. To examine the importance of palmitoylation sites Cys3 and Cys5 in Lck, one or both of these residues was mutated to serine to create mutants S3, S5, and S3,5, respectively. Immunofluorescence and confocal microscopy of COS-7 cells transfected with these constructs showed that while S5 and S3 localized to the plasma membrane, S3,5 was localized to the cytoplasm, suggesting that palmitoylation at at least one site is essential for membrane localization. Sucrose gradient based fractionation of these mutants expressed in COS-7 cells showed that while S5 localized to GEMs in similar fashion to the wild type, GEM localization of S3 was severely inhibited. Expression of these mutants in Lck-negative JCaM1 cells showed that although S5 reconstituted activation of nuclear factor NFAT as per the wild type, S3 expression failed to do so. These results suggest that Cys3 of Lck plays a more important role than Cys5 in GEM localization and T cell activation. Additionally, it was found that the degree of T cell function recovery is positively correlated with the degree of Lck expression in GEMs.

Animals↗

PubMed2001

Influence of cataract surgery on automated perimetry in patients with glaucoma.

PURPOSE: To investigate the influence of cataract surgery on automated perimetry in patients with glaucoma. DESIGN: Interventional case series. METHODS: A total of 105 eyes of 105 consecutive patients with glaucoma who were scheduled for cataract surgery underwent Humphrey static threshold testing (30-2 program) before and at 1 month after surgery. These eyes were divided into two groups based on the presence of absolute or near absolute (dense) scotomata before surgery (minimum threshold value 5 decibels or less). Changes in numbers of the dense scotomata, mean deviation, pattern standard deviation, and corrected pattern standard deviation were analyzed. RESULTS: No significant changes were found in the numbers of dense scotomata and central dense scotomata within five-degree visual fields between before and after surgery. In the group with dense scotomata, the mean pattern standard deviation and corrected pattern standard deviation worsened significantly after surgery, whereas the mean deviation improved significantly. However, in the group without dense scotomata, the pattern standard deviation and corrected pattern standard deviation showed a slight improvement, whereas the mean deviation improved significantly. When the central scotoma was present in two or fewer meridians before surgery, the mean visual acuity after surgery reached 20/25 and was better than that in patients with three or four central scotomata (P =.0014). CONCLUSIONS: Cataract does not produce a dense scotoma on automated perimetry. However, because it does produce relative scotomata, actual glaucomatous visual field defects may be hidden to some extent. When a central dense scotoma is present before surgery in two or fewer meridians, the patient may well achieve substantial improvement in postoperative visual acuity.

Adult↗

PubMed2001

Correlation between pupillary size and intraocular lens decentration and visual acuity of a zonal-progressive multifocal lens and a monofocal lens.

OBJECTIVE: To see whether there is a correlation between pupillary area or intraocular lens (IOL) decentration and tilt and the visual acuity (VA) at all distances for eyes with a zonal-progressive multifocal IOL or a monofocal IOL. DESIGN: Comparative, nonrandomized, interventional study. PARTICIPANTS: Fifty-five eyes of 55 patients undergoing five-zone refractive multifocal IOL implantation and 55 eyes of 55 age-matched patients undergoing monofocal IOL implantation. INTERVENTION: All eyes underwent phacoemulsification and IOL implantation. MAIN OUTCOME MEASURES: The VAs from far to near distances were examined using an all-distance vision tester at 1 month after surgery. The pupillary area was also measured using an infrared pupillometer, and the degrees of IOL decentration and tilt were measured using a Scheimpflug videophotography system. Univariate associations between VA and the pupillary area and IOL decentration or tilt were evaluated statistically. RESULTS: The mean intermediate VAs and contrast sensitivities at all spatial frequencies in the multifocal IOL group were worse than those in the monofocal IOL group, whereas near VA was better in the multifocal group. In the multifocal group, smaller pupillary area was associated significantly with worse near logarithm of the minimum angle of resolution (LogMAR) VA (r = 0.636), but not with far or intermediate VAs. When pupil diameter was 4.5 mm or greater, near mean VA reached 20/63. A greater degree of IOL decentration was associated significantly with worse far and intermediate LogMAR VAs (r = 0.460 at 5.0 m and 0.527 at 1.0 m) but not with near VA. When decentration was 0.9 mm or greater, distance mean VA did not reach 20/32. However, the correlation between tilt and VA was not statistically significant. In the monofocal group, no significant correlation was found between pupillary area, IOL decentration, or tilt and the VA. CONCLUSIONS: Smaller pupil size is correlated significantly with worse near VA, whereas greater decentration is correlated with worse distance and intermediate VA in eyes with refractive multifocal IOLs. Specifically, a pupil diameter of less than 4.5 mm cannot provide useful near VA, and decentration of 0.9 mm is the maximum allowable limit for adequate distance VA. However, pupil size and IOL decentration do not influence VA in eyes with monofocal IOLs.

Adult↗

PubMed2001

Changes in posterior capsule opacification after poly(methyl methacrylate), silicone, and acrylic intraocular lens implantation.

PURPOSE: To prospectively evaluate the progression of posterior capsule opacification (PCO) after poly(methyl methacrylate) (PMMA), silicone, and acrylic intraocular lens (IOL) implantation. SETTING: Hayashi Eye Hospital, Fukuoka, Japan. METHODS: Three hundred eyes of 300 patients scheduled to have IOL implantation were initially randomized into 3 groups based on IOL type: PMMA, silicone, or acrylic. Of the 300 eyes, 269 completed the follow-up. The PCO density in these eyes was measured 1 week and 3, 6, 12, 18, and 24 months postoperatively using special computer software developed for the Scheimpflug videophotography system. Visual acuity and the incidence of neodymium:YAG (Nd:YAG) laser capsulotomy were also examined. RESULTS: Three months postoperatively and later, the mean PCO value in the PMMA group increased significantly (P <.0001); the increase in the silicone and acrylic groups was not significant. The PCO value in the PMMA group was significantly greater than in the silicone or acrylic group (P <.0001). The PCO value in the acrylic group was slightly less than in the silicone group at 18 and 24 months, but the difference was marginal. The survival rate not requiring Nd:YAG capsulotomy was least in the PMMA group, followed by the silicone and acrylic groups in that order (P <.0001). The mean logMAR visual acuity in the PMMA group increased postoperatively and was worse than in the silicone or acrylic group. CONCLUSIONS: The degree of PCO after PMMA IOL implantation progressed significantly with time, while the progression after silicone and acrylic IOL implantation was slight. Therefore, PCO in eyes with a PMMA IOL was significantly more extensive than in those with a silicone or acrylic IOL and resulted in marked impairment of visual acuity.

Acrylic Resins↗

PubMed2001

Effect of cataract surgery on intraocular pressure control in glaucoma patients.

PURPOSE: To examine the effect of cataract surgery on intraocular pressure (IOP) control in eyes with angle-closure glaucoma (ACG) and open-angle glaucoma (OAG). SETTING: Hayashi Eye Hospital, Fukuoka, Japan. METHODS: This study included 74 eyes with ACG and 68 eyes with OAG having cataract surgery. The IOP was measured and the number of glaucoma medications recorded preoperatively, 1 month postoperatively, and then every 3 months. The IOP control in the 2 groups was compared using survival analysis, with failure criteria being an IOP greater than 21 mm Hg, addition of medications, or the need for additional glaucoma surgery. RESULTS: The mean IOP and number of medications decreased significantly after surgery in both groups (P <.0001). However, the mean decrease in IOP and percentage of IOP reduction in the ACG group were greater than in the OAG group, and fewer medications were required in the ACG group. The cumulative survival probability of IOP control at 24 months was 91.9% in the ACG group and 72.1% in the OAG group. The survival curve in the ACG group was significantly better than in the OAG group (P =.0012). The IOP was controlled without medication in 30 eyes (40.5%) in the ACG group and 13 (19.1%) in the OAG group; the difference between groups was significant (P =.0055). CONCLUSIONS: Cataract surgery substantially reduced IOP and the number of medications required for IOP control in glaucomatous eyes. Specifically, cataract extraction normalized the IOP in most eyes with ACG.

Aged↗

PubMed2001

PCR detection of 5 putative periodontal pathogens in dental plaque samples from children 2 to 12 years of age.

BACKGROUND, AIMS: The purpose of this study was to detect the presence of Prevotella intermedia, P. nigrescens, Bacteroides forsythus, Treponema denticola, and Campylobacter rectus in plaque samples from 119 children, collected from their toothbrushes using a polymerase chain reaction (PCR). METHOD: The subjects were 24, 83, and 12 children with healthy gingiva, gingivitis, and periodontitis, respectively, ranging in age from 2-12 years old. Plaque samples were collected from all erupted teeth sites using a sterile toothbrush. The mean concentration of DNA recovered from the plaque samples was approximately 640 microg/ml, which was deemed sufficient for performing a PCR-based survey. RESULTS: The prevalence by PCR in healthy, gingivitis, and periodontitis subjects was 0.0%, 6.0% and 25.0% for P. intermedia, 45.8%, 79.5% and 50.0% for P. nigrescens, 33.3%, 63.9% and 58.3% for B. forsythus, 0.0%, 18.1% and 16.7% for T. denticola, and 100% in duplicate for C. rectus, respectively. CONCLUSION: Our survey indicated that P. intermedia and T. denticola were more associated with periodontal diseases, B. forsythus and P. nigrescens had a moderate prevalence in all clinical groups, while C. rectus were the most commonly detected species in the oral cavities of children suggesting establishment in their early years.

Age Factors↗

PubMed2001

Leptospiral lipopolysaccharide activates cells through a TLR2-dependent mechanism.

Leptospira interrogans are zoonotic pathogens that have been linked to a recent increased incidence of morbidity and mortality in highly populated tropical urban centers. They are unique among invasive spirochetes in that they contain outer membrane lipopolysaccharide (LPS) as well as lipoproteins. Here we show that both these leptospiral outer membrane constituents activate macrophages through CD14 and the Toll-like receptor 2 (TLR2). Conversely, it seems that TLR4, a central component for recognition of Gram-negative LPS, is not involved in cellular responses to L. interrogans. We also show that for intact L. interrogans, it is LPS, not lipoprotein, that constitutes the predominant signaling component for macrophages through a TLR2 pathway. These data provide a basis for understanding the innate immune response caused by leptospirosis and demonstrate a new ligand specificity for TLR2.

Animals↗

PubMed2001

Purification and characterization of Delta(1)-pyrroline-5-carboxylate reductase isoenzymes, indicating differential distribution in spinach (Spinacia oleracea L.) leaves.

Delta(1)-Pyrroline-5-carboxylate reductase (P5CR) (EC 1.5.1.2. L-proline: NAD(P)-5-oxidoreductase), the second enzyme in the proline biosynthetic pathway, was purified from spinach (Spinacia oleracea L.) leaves. Following ammonium sulfate fractionation, purification was performed by several chromatographic methods: Blue Cellulofine, DEAE-TOYOPEARL, Sephacryl S-300 HR, and POROS QE/M. Two isoenzymes resolved by anion exchange chromatography were designated P5CR-1 and P5CR-2. Only P5CR-2 was purified from the intact chloroplasts, indicating differential distribution of the isoenzymes. P5CR isoenzymes, P5CR-1 and P5CR-2, are a homopolymer with an apparent molecular mass of 310 kDa, consisting of 10 to 12 subunits of about 28.5 kDa. P5CR-1 and P5CR-2 showed K(m) values of 9 and 19 microM for NADPH and values of 0.122 and 0.162 mM for Delta(1)-pyrroline-5-carboxylate (P5C), respectively. We decided partial amino acid sequences of P5CR-1 which showed the 70 to 80% homology to the deduced amino acid sequences of several plant P5CR cDNAs. Both isoenzymes had much lower affinity for NADH than for NADPH and were inhibited by free ATP and Mg(2+) ion. The inhibition was partially mitigated when ATP and Mg(2+) were added simultaneously to the reaction mixture. Cations at high concentration were inhibitory to P5CR activity. Interestingly, P5CR-2 was more stable to heat treatment at 40 degrees C than P5CR-1.

Acrylic Resins↗

PubMed2001

PCR detection of Capnocytophaga species in dental plaque samples from children aged 2 to 12 years.

The purpose of this study was to detect the presence of Capnocytophaga sputigena, C. ochracea, and C. gingivalis in plaque samples from the toothbrushes of 122 children, using a polymerase chain reaction (PCR) method. The subjects were 25, 85, and 12 children with healthy gingiva, gingivitis, and periodontitis, respectively, ranging in age from 2-12 years old. Plaque samples were collected from all erupted tooth sites using a sterile toothbrush. The mean amount of DNA recovered from the samples was approximately 19.3 microg, which was deemed sufficient for performing a PCR-based survey. C. sputigena prevalence in healthy, gingivitis, and periodontitis subjects was 48.0%, 36.5% and 25.0%, respectively, that for C. ochracea was 100%, 89.4%, and 50.0%, respectively, and that for C. gingivalis was 96.0%, 84.7%, and 75.0%, respectively. The lowest age of positive subjects was approximately 2 years. Our results showed that C. sputigena was moderately prevalent, whereas C. ochracea and C. gingivalis were commonly detected in the oral cavities of the tested children, suggesting that all of these species become established in the early years.

Bacterial Typing Techniques↗

PubMed2001

Anterior capsule contraction and intraocular lens decentration and tilt after hydrogel lens implantation.

AIM: To prospectively investigate changes in the area of the anterior capsule opening, and intraocular lens (IOL) decentration and tilt after implantation of a hydrogel IOL. METHODS: 100 patients underwent implantation of a hydrogel IOL in one eye and an acrylic IOL implantation in the opposite eye. The area of the anterior capsule opening, and the degree of IOL decentration and tilt were measured using the Scheimpflug videophotography system at 3 days, and at 1, 3, and 6 months postoperatively. RESULTS: The mean anterior capsule opening area decreased significantly in both groups. At 6 months postoperatively, the area in the hydrogel group was significantly smaller than that in the acrylic group. The mean percentage of the area reduction in the hydrogel group was also significantly greater than that in the acrylic group, being 16.9% in the hydrogel group and 8.8% in the acrylic group. In contrast, IOL decentration and tilt did not progress in either group. No significant differences were found in the degree of IOL decentration and tilt throughout the follow up period. CONCLUSIONS: Contraction of the anterior capsule opening was more extensive with the hydrogel IOL than with the acrylic IOL, but the degree of IOL decentration and tilt were similar for the two types of lenses studied.

Acrylic Resins↗

PubMed2001

Inositol-1,4,5-trisphosphate-binding proteins controlling the phototransduction cascade of invertebrate visual cells.

The main phototransduction cascade in invertebrate visual cells involves the turnover of phosphatidylinositol, an important biochemical mechanism common to many signal-transduction systems. Light-activated rhodopsin stimulates guanine nucleotide exchange on the Gq class of G-protein, which activates phospholipase C to hydrolyze phosphatidylinositol 4,5-bisphosphate to inositol-1,4,5-trisphosphate and diacylglycerol. Subsequently, inositol-1,4,5-trisphosphate-binding proteins continue the signal cascade. Here, we report on the first inositol-1,4,5-trisphosphate-binding proteins demonstrated in an invertebrate visual system with our investigation of the photosensitive rhabdoms of squid. We screened the ability of proteins to interact with inositol-1,4,5-trisphosphate by affinity column chromatography with an inositol-1,4,5-trisphosphate analogue. We detected an inositol-1,4,5-trisphosphate-binding affinity in phospholipase C, receptor kinase and five other proteins in the cytosolic fraction and, surprisingly, rhodopsin in the membrane fraction. A binding assay with (3)H-labelled inositol-1,4,5-trisphosphate demonstrated the inositol-1,4,5-trisphosphate affinity of each of the purified proteins. Since rhodopsin, receptor kinase and phospholipase C are involved upstream of phosphatidylinositol turnover in the signal cascade, our result suggests that phosphatidylinositol turnover is important in feedback pathways in the signalling system.

Animals↗

PubMed2001

[Chronobiological case study on cardiac responses during aerobic exercise].

50% HR reserve aerobic exercise on a bicycle ergometer was carried out for 20 min each time by a slightly obese male aged 70 years old from October, 1998 to August, 2000 at 8:00 AM (Mor. Ex.) or 17:00 PM (Even. Ex.). The same exercise was carried out at random times during the day from January to March, 2000. The following five indicators were measured: the heart rate in the first minute of exercise, the time until the attainment of the target heart rate, the loaded power at the time of attaining the target heart rate, the energy consumption during exercise, and RPE on the Borg Scale. The effects of Jet lag were also observed three times during that period. The following results were obtained. I. The changes in indicators with the progress of exercise; 1. The heart rate after the first minute of exercise was increased in the Mor. Ex. 2. The time until attainment of the target heart rate was shortened in the Mor. Ex., but it was unchanged in the Even. Ex. 3. The loaded power at the time when the target heart rate was attained decreased in the Mor. Ex., but it was unchanged in the Even. Ex. 4. Energy consumption decreased in the Mor. Ex., and increased in the Even. Ex. 5. The RPE decreased in the Mor. Ex., but increased in the Even. Ex. The abovementioned changes were not due to seasonal variations. II. The effects of exercise became stable at and after six months in the Mor. Ex., and nine months in the Even. Ex. III. The differences between Mor. Ex., and Even. Ex. in all the indicators were significant. IV. The coefficient of correlation between the energy consumption and heart rate at the end of the first minute of exercise was significantly inverse. V. Energy consumption during exercise increased from 8:00 AM and left the range of morning response at 9:30 AM and entered the range of evening response on and after 11:00 AM. It decreased for a short time after lunch. VI. The effect of Jet lag was observed as a lowering of energy consumption during exercise in the Even. Ex., and two or four days were required for recovery.

Aged↗

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↗