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Biomedical subjects

R H Lee

Publications and source records attributed to R H Lee.

At least 19 recordsLinked to original sources
PubMed1992

Regulation of retinal cGMP cascade by phosducin in bovine rod photoreceptor cells. Interaction of phosducin and transducin.

Photoexcitation of retinal rod photoreceptor cells involves the activation of cGMP enzyme cascade in which sequential activation of rhodopsin, transducin, and the cGMP phosphodiesterase in the rod outer segment constitutes the signal amplification mechanism. Phosducin, a 33-kDa phosphoprotein, has been shown to form a tight complex with the T beta gamma subunit of transducin. In this study, we examined the interaction of phosducin-T beta gamma and the possible regulatory role of phosducin on the cGMP cascade. Addition of phosducin to photolyzed rod outer segment (ROS) membrane reduced the GTP hydrolysis activity of transducin as well as the subsequent activation of the cGMP phosphodiesterase. Phosducin also inhibited the pertussis toxin-catalyzed ADP-ribosylation of transducin, indicating that the interaction between the T alpha and T beta gamma subunits of transducin was interrupted upon binding of phosducin. The inhibitory effects of phosducin were reversed by the addition of exogenous T beta gamma. These results suggest that phosducin is capable of regulating the amount of T beta gamma available to interact with T alpha to form the active transducin complex and thereby functions as a negative regulator of the cGMP cascade. The phosducin-induced alteration of the subunit organization of transducin was examined by chemical cross-linking method using para-phenyl dimaleimide as cross-linker. It was found that the cross-linking among T alpha and T beta gamma was blocked in the presence of phosducin. This result implies that T beta gamma may undergo a conformational change upon phosducin binding which leads to the release of T alpha. Since phosducin is a soluble protein, the interaction with transducin only occurs when transducin is dissociated from ROS disc membrane. Indeed, phosducin failed to dissociate membrane-bound transducin and did not inhibit the initial cycle of transducin activation as measured by the presteady state GTP hydrolysis. However, phosducin interacts effectively with transducin released into solution after the initial activation and blocks the re-binding of T alpha. T beta gamma to ROS membrane by forming a tight complex with T beta gamma. This interaction may play an important role in regulating the turnover of the cGMP cascade in photoreceptor cells.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1992

A third form of the G protein beta subunit. 2. Purification and biochemical properties.

Visual excitation in cones is thought to involve a cone-specific G protein (cone transducin) that transduces the light signal detected by the cone visual pigment into an increase in the enzymatic activity of a cGMP phosphodiesterase. In the preceding paper, we have shown that the G beta 3 isoform of G proteins is specifically localized in bovine cone photoreceptors and proposed that it might be a component of cone transducin. We reported here the purification from bovine retinal extract of a cone-specific T beta 3 gamma complex (where T is transducin), which is composed of a G beta 3 subunit and an immunochemically distinct G gamma subunit. Our purification of this complex is based on a two-stage procedure; the first stage consists of a series of column chromatographies that yield a mixture of purified T beta gamma substantially enriched in T beta 3 gamma, and the second stage involves the removal of all of the rod-specific T beta 1 gamma from the mixture using an affinity column of immobilized monoclonal antibodies directed against the rod T gamma subunit of transducin. Using this procedure, we were able to obtain sufficient amounts of T beta 1 gamma and T beta 3 gamma to begin a comparative study of their properties. We showed that T beta 3 gamma is distinguishable from T beta 1 gamma by isoelectric focusing under nondenaturing conditions. The G beta 3 polypeptide of T beta 3 gamma also migrates slightly slower than the G beta 1 polypeptide of T beta 1 gamma on denaturing polyacrylamide gels. Analysis of the interactions of T beta 3 gamma with other retinal proteins indicated that it has a lower affinity for the T alpha subunit of rod transducin but appears to complex with a phosducin-like protein. The differences in the intrinsic biochemical properties of T beta 3 gamma as compared to T beta 1 gamma may partially account for the lower light sensitivity of cones.

Amino Acid Sequence↗
PubMed1992

A third form of the G protein beta subunit. 1. Immunochemical identification and localization to cone photoreceptors.

Vertebrate retinal cones play a major role in both photopic vision and color perception. Although the molecular mechanism of visual excitation in the cone is not as well understood as in the rod, it is generally thought to involve a cone-specific G protein (cone transducin) that couples the cone visual pigment to a cGMP phosphodiesterase. Like all other G proteins, cone transducin is most likely a heterotrimer consisting of G alpha, G beta, and G gamma subunits. A G alpha subunit of cone transducin has been localized to the outer segment of bovine cones, but its associated G beta and G gamma subunits are unknown. To identify the G beta subunit involved in the phototransduction process of cones, we have developed a panel of antipeptide antisera against the most diverse region of the amino acid sequences encoded by G beta 1, G beta 2, and G beta 3 cDNAs and used them to determine the distribution of the G beta isoforms in different retinal preparations. We found that the G beta 3 subunit is present in bovine retinal transducin and phosducin-T beta gamma complex preparations which were previously thought to contain only G beta 1. Analysis of its subcellular distribution indicated that G beta 3 is predominantly cytoplasmic. Immunocytochemical staining of bovine retinal sections with the anti-G beta 3 antiserum further revealed a specific localization of G beta 3 in cones but not in rods. In contrast, anti-G beta 1 antiserum stained only the rods. These results suggest that G beta 3 is the G beta subunit of cone transducin and confirms the proposition that rods and cones utilize distinct signaling proteins for phototransduction.

Amino Acid Sequence↗
PubMed1992

Marine sponge polyketide inhibitors of protein tyrosine kinase.

The marine polyketide natural product, halenaquinone, was shown to be an irreversible inhibitor of pp60v-src, the oncogenic protein tyrosine kinase encoded by the Rous sarcoma virus. This compound had an IC50 of approximately 1.5 microM against pp60v-src and also inhibited the ligand-stimulated kinase activity of the human epidermal growth factor receptor with an IC50 of approximately 19 microM. Halenaquinone blocked the proliferation of a number of cultured cell lines, including several transformed by oncogenic protein tyrosine kinases. Halenaquinol, xestoquinone, halenaquinol sulfate, and several simple synthetic quinone analogs were also shown to inhibit pp60v-src.

Animals↗
PubMed1992

Photoreceptors of the retina and pinealocytes of the pineal gland share common components of signal transduction.

Light absorbed by retinal photoreceptors triggers a cascade of reactions that initiate cGMP hydrolysis, cation channel closure and membrane hyperpolarization. Down-regulation of the cascade involves additional proteins that interfere with amplification along the cascade. Pinealocytes are activated by norepinephrine during the dark phase of the day/night cycle. Mature pinealocytes of the mammalian pineal express the known photoreceptor proteins that are implicated in down-regulation of the visual cascade, but the cascade components that produce cGMP hydrolysis and membrane hyperpolarization are absent. Pinealocytes accumulate cyclic AMP minimally when norepinephrine activates their beta adrenergic receptors alone, but the response is potentiated by the simultaneous activation of their alpha-1 adrenergic receptors. A model is proposed whereby phosducin, a phosphoprotein that binds the beta,gamma subunit of G-proteins, could modulate the synthesis of cyclic AMP by buffering the amount of beta,gamma G-protein subunits that are available for activating adenylate cyclase.

Animals↗
PubMed1992

Induction of experimental autoimmune uveitis by the retinal photoreceptor cell protein, phosducin.

Experimental autoimmune uveitis (EAU) and experimental autoimmune pinealitis (EAP) are CD4+ T cell mediated inflammatory diseases of the retina and uveal tract of the eye and the pineal gland respectively. They can be induced in experimental animals by immunization with several well characterized retinal autoantigens. We induced a mild to moderate EAU and EAP in Lewis rats by immunization with phosducin, a 33K retinal phosphoprotein which is involved in the phototransduction of vision. In contrast to the severe EAU induced by other retinal antigens like S-antigen (SAg) or interstitial retinoid binding protein (IRBP), the clinical disease was late in onset, low grade in severity and predominantly affected the posterior segment of the eye. Our study demonstrates that another photoreceptor cell protein, phosducin, is capable of eliciting EAU and EAP.

Animals↗
PubMed1991

Rat pineal gland phosducin: cDNA isolation, nucleotide sequence, and chromosomal assignment in the mouse.

The pineal gland contains a soluble phosphoprotein, phosducin, which is homologous to that of retinal photoreceptors. Phosducin has been shown to bind the beta, gamma subunits of the retinal G-protein transducin. Retinal phosducin has been cloned and now we report a rat pineal cDNA encoding phosducin. A 1217-nucleotide cDNA was isolated from a rat pineal library by DNA-DNA hybridization with a polymerase chain reaction-amplified cDNA of bovine retina mRNA for phosducin. Northern blot analysis demonstrates that the mRNA for phosducin is approximately 1.3 kb in both rat pineal and rat retina. The translated mRNA from rat pineal encodes a protein with 246 amino acids, compared to the 245 amino acids of bovine retina phosducin. The predicted molecular weight of rat pineal phosducin is 28,201. Immunoblot analysis with affinity-purified antibodies against bovine retina phosducin identify a single immunoreactive protein of approximately 33 kDa in both rat retina and rat pineal. The amino acid sequence of rat pineal phosducin is homologous to that of bovine retina phosducin, revealing 89% identity and another 5.7% similarity. Both rat pineal and bovine retina phosducins are acidic proteins with pIs of 4.3 and 4.5, respectively. The translated protein lacks hydrophobic domains that would suggest an integral membrane protein. Rat pineal phosducin has a single consensus phosphorylation domain for protein kinase A that is nearly identical to that of retinal phosducin, which is phosphorylated by protein kinase A in situ. Rat phosducin also contains three potential phosphorylation domains for protein kinase C and nine for casein kinase II as well as a predicted site for N-glycosylation. The cDNA encoding phosducin was used to localize the gene within a linkage group to a large segment of mouse chromosome 1 in a conserved region with the long arm of human chromosome 1 with a panel of DNA samples from an interspecific cross. In keeping with a proposed role of retinal phosducin in down-regulation of the photo-transduction cascade, a modulatory role in signal transduction is proposed for pineal phosducin.

Amino Acid Sequence↗
PubMed1991

Family structure and physicians' hours in large, multispecialty groups.

There is a consensus that male physicians work more hours than female physicians, but no consensus on how or why female physicians limit hours. Drawing on a new national survey of physicians in large, multispecialty groups, we conclude that much of the hours differential is due to differences in nonpractice incomes and is likely to be permanent. However, due to reductions in family responsibilities for women and increases in nonpractice incomes for men, the differential should narrow somewhat as the current cohort of physicians ages. We also infer that the link between hours and practice choices may well account for much of the diversity of the literature.

Family↗
PubMed1990

Amino acid and cDNA sequence of bovine phosducin, a soluble phosphoprotein from photoreceptor cells.

Vertebrate photoreceptor cells contain a soluble phosphoprotein, phosducin, which complexes with the beta, gamma subunits of the GTP-binding protein, transducin. Light-induced changes in cyclic nucleotide levels modulate the phosphorylation of phosducin by protein kinase A. The complete amino acid sequence of purified phosducin from bovine retinas was determined by Edman degradation from overlapping polypeptides derived from enzymatic digestion by trypsin and Staphylococcus aureus V8 protease or from chemical degradation by cyanogen bromide. Excluding the unidentified group which blocks the NH2 terminus, phosducin contains 245 amino acids with a calculated molecular weight of 28,185 and isoelectric point of pH 4.5. Phosducin is enriched with acidic and sulfur-containing amino acids, having 32 glutamic acid, 16 aspartic acid, 9 methionine, and 5 cysteine residues. It also contains 24 serine and 8 threonine residues, of which only serine 73 is located within a consensus phosphorylation sequence (-RKMS(P)QV-) for cyclic nucleotide-dependent protein kinase. Secondary structure analysis predicts the presence of 62% alpha-helix, 22% beta-sheet, and 16% random coil, with eight turns. Computer-aided searches of protein data banks revealed no apparent homology to any sequenced protein except that coded by a MEKA cDNA clone (Kuo, C-H., Akiyama, M., and Miki, N. (1989) Mol. Brain Res. 6, 1-10) which deviates from the confirmed phosducin sequence in the last 15 amino acids. Sequence analysis of a cDNA clone for bovine retinal phosducin confirmed that the MEKA clone deviation resulted from an unidentified cDNA guanosine nucleotide, a shifted reading frame and a premature stop codon.

Amino Acid Sequence↗
PubMed1990

Protein kinase A phosphorylates retinal phosducin on serine 73 in situ.

Photoreceptors of vertebrate retinas contain a 33,000-dalton phosphoprotein, phosducin, which complexes with the beta, gamma subunits of the photoreceptor G-protein (guanine nucleotide-binding protein), transducin. In situ, the retinal content of phosphorylated phosducin is modulated by light in conjunction with light-triggered changes in intracellular cyclic nucleotide concentration. In vitro, phosducin is phosphorylated by either exogenous or endogenous protein kinase A. 32P-Labeled rat retina phosducin was isolated by immunoprecipitation either after phosphorylation by protein kinase A in the presence of [gamma-32P]ATP or after incubation of retinas in darkness with 32Pi. In either case, phosphoamino acid analysis showed that greater than 98% of 32P was linked to serine, with less than 2% to threonine. Two-dimensional peptide mapping showed that [32P]phosphoserine was associated with the same characteristic set of tryptic peptides. Furthermore, Cleveland peptide analysis using four different proteases showed that either sample exhibited identical patterns of phosphopeptides which were characteristic of the protease used. Identical phosphopeptide maps were also obtained from 32P-labeled bovine retina phosducin, indicating that the serine phosphorylation site for protein kinase A is conserved between rat and bovine. Edman degradation of phosphopeptides derived from 32P-labeled bovine phosducin showed that radioactive phosphate was incorporated into serine residue 73 which is located within a consensus phosphorylation sequence for protein kinase A (-R-K-M-S73(P)-). These observations are uniformly in agreement with protein kinase A being the endogenous kinase that phosphorylates phosducin in vivo.

Adenosine Triphosphate↗
PubMed1990

Retinal accumulation of the phosducin/T beta gamma and transducin complexes in developing normal mice and in mice and dogs with inherited retinal degeneration.

Rod photoreceptors of mammalian retinas contain a 33-kDa phosphoprotein, phosducin, which complexes with the beta, gamma-subunits of transducin (T beta gamma). The level of phosducin phosphorylation is modulated by light, suggesting that the phosducin/T beta gamma complex has a pivotal role in light-regulated events that occur in photoreceptors. We have investigated, in developing mouse retinas, the age at which the complex is first detected and the subsequent accumulation of the phosducin/T beta gamma complex during postnatal life. Western blot analysis detected immunoreactivity both for phosducin and T beta in retinal homogenates of 3-day-old mice. Thereafter, the level of immunoreactivity for both proteins increased steadily, to reach adult levels in the next 2 postnatal weeks. Gel filtration analysis of extracts from immature mouse retina showed that phosducin and T beta co-eluted, like the phosducin/T beta gamma complex of adult retina, as a 77-kDa complex, indicating that the phosducin/T beta gamma complex is formed when photoreceptors first synthesize the components of the complex. While the levels of the phosducin/T beta gamma complex increased steadily during the first 2 postnatal weeks, the subunits of transducin complex, T alpha together with additional amounts of T beta gamma, only started to appear around the 7-9th postnatal day, and the level of transducin complex increased sharply at 11-14 days to reach adult levels that are similar to those of phosducin/T beta gamma complex.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

Cyclic GMP and photoreceptor function.

A single photon can be detected by a rod photoreceptor cell. The absorption of light by rhodopsin triggers a cascade of reactions that amplifies the photon signal and results in ion channel closure with hyperpolarization of the rod photoreceptor cell. Light-induced conformational changes in rhodopsin facilitate the binding of a guanosine nucleotide-binding protein, transducin, which then undergoes a GTP-GDP exchange reaction and dissociation of the transducin complex. A subunit of transducin then activates a phosphodiesterase complex that hydrolyzes cyclic GMP. In darkness, cyclic GMP binds to cation channels of the photoreceptor plasma membrane, maintaining them in an open configuration. The light-induced reduction in cyclic GMP concentration dissociates the bound cyclic GMP, resulting in channel closure and hyperpolarization. Down-regulation of the cascade involves other proteins that block the interaction of transducin with rhodopsin and another protein that may interfere with transducin recycling. Cone photoreceptors possess a light-activated cascade that follows the rod format, but it is composed of proteins that are homologous to those of rod photoreceptors. Phototransduction in invertebrate photoreceptors uses rhodopsin to activate a cascade that uses phosphoinositides and calcium ion to regulate membrane polarization.

Antigens↗
PubMed1988

Evaluation of left ventricular contractility indexes for the detection of symptomatic and silent myocardial ischemia.

This study evaluated the role of left ventricular (LV) ejection fraction and systolic blood pressure (BP) to end-systolic volume ratio to detect symptomatic and silent myocardial ischemia. The sensitivity and diagnostic accuracy of these contractility indexes were compared with angina and ST depression during exercise. Thirty consecutive patients referred for chest pain performed symptom-limited bicycle exercise and had coronary angiography within 3 months. Twenty-two had angiographically significant coronary artery disease and 8 had normal coronary anatomy. Systolic BP was measured by sphygmomanometry; LV ejection fraction and end-systolic volume were obtained by nuclear ventriculography. Normal values for contractility indexes were defined as LV ejection fraction greater than 52% at rest and increment of greater than or equal to 5% during exercise, and systolic BP to end-systolic volume ratio greater than 2.2 mm Hg/ml at rest and greater than 3.0 mm Hg/ml during exercise. The sensitivity of systolic BP to end-systolic volume ratio to identify patients with coronary artery disease at rest was 71 vs 33% for LV ejection fraction. During exercise, each contractility index had a sensitivity of 95% and there was a combined sensitivity of 100%. This compares with 71% for ST depression and 48% for exercise-induced angina. Thus, 52% had no angina and 36% of them were also silent by electrocardiography. Among the patients with symptomatic ischemia, 20% had no ST-segment depression. Measurement of contractility indexes enhanced the detection of silent myocardial ischemia and provided information on LV function vital to prognosis and management of patients with coronary artery disease.

Adult↗
PubMed1988

The photoreceptor-specific 33 kDa phosphoprotein of mammalian retina: generation of monospecific antibodies and localization by immunocytochemistry.

The distribution in mouse retina of a 33,000 Da phosphoprotein (33 kDa) that complexes with the beta/gamma subunits of transducin (T beta gamma) and undergoes light-induced dephosphorylation was determined by immunocytochemistry. An antiserum containing antibodies for the 33 kDa protein and beta-transducin of mouse and bovine retinas was generated against the purified 33 kDa-T beta gamma complex from bovine retina. The antiserum reacts with beta-transducin derived from either 33 kDa-T beta gamma complex or transducin complex (T alpha beta gamma), but not with the alpha- or gamma-transducin. It also reacts with both the phosphorylated and unphosphorylated form of the 33 kDa-T beta gamma complex. Antibodies, monospecific for the 33 kDa and beta-transducin subunits respectively, were purified from the antiserum by immunoadsorption and used in immunocytochemical analysis of the respective antigens. The 33 kDa protein was found to be associated exclusively with the photoreceptor cells of the retinas, with the most intense staining in the inner and outer segments' layers and lighter staining in the synaptic terminal layers. beta-Transducin also is found in the photoreceptors, but some T beta immunoreactivity exists within the inner plexiform layer. The specific localization of the 33 kDa protein together with its light-modulated phosphorylation suggest that the 33 kDa-T beta gamma complex is involved in light-regulated activities of the rod photoreceptor cells.

Animals↗
PubMed1988

Characterization of a phosphodiesterase-immunoreactive polypeptide from rod photoreceptors of developing rd mouse retinas.

In the inherited retinal degeneration of rd mice, cyclic GMP accumulates in affected rod photoreceptors prior to their degeneration. A deficiency in the activity of the visual cell phosphodiesterase apparently results in the accumulation of cyclic GMP. The cyclic GMP phosphodiesterase (PDE) of normal mouse photoreceptors is a heteromeric protein complex of about 170 kDa, consisting of the alpha beta catalytic unit and the gamma inhibitory unit. The isolated complex has low enzyme activity but it can be activated by incubation with histone. Affinity-purified polyclonal antibodies against the PDE complex of bovine rod outer segments were prepared and used to identify in retinas of both normal and rd mice PDE-immunoreactive polypeptides which comigrated on SDS-polyacrylamide gels with the large subunits (88 kDa) of the normal PDE complex. During development of normal retinas, the 88 kDa immunoreactive component of the PDE complex were detected by day 7, with immunoreactivity increasing throughout the second postnatal week. In rd retinas, the 88 kDa immunoreactivity increased after 9 postnatal days, decreased during rod photoreceptor degeneration, and was undetectable in mature rd retinas. Under nondenaturing conditions, the PDE-immunoreactive polypeptide of rd retinas sedimented on sucrose gradients with a sedimentation coefficient of 5.6S and an apparent molecular mass of about 105 kDa; no associated histone-activated PDE activity was detected. These findings show that PDE-immunoreactive polypeptides are synthesized in immature rd photoreceptors and that the PDE-immunoreactive polypeptides fail to form a PDE complex which is comparable to that of normal photoreceptors.

Animals↗
PubMed1988

Tic disorders of childhood.

Tic disorders may be classified as simple tics, chronic motor tics or Tourette syndrome, the most severe of the three types. Tourette syndrome is rather uncommon and is often misdiagnosed. Tic disorders of childhood probably have an organic etiology and a genetic component. An increased incidence of obsessive-compulsive behavior, attention deficit disorder and other abnormalities have been reported in Tourette patients. Haloperidol is often useful in controlling severe tic symptoms, while stimulant drugs may worsen the symptoms.

Attention Deficit Disorder with Hyperactivity↗