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At least 1,225 records · Page 68Linked to original sources
PubMed1992

Immunoglobulin-mediated phagocytosis by human monocytes requires protein kinase C activation. Evidence for protein kinase C translocation to phagosomes.

This study has investigated the role of protein kinase C (PKC) activation in IgG-mediated phagocytosis by human monocytes. Incubation of monocytes with IgG-opsonized targets increased membrane-associated PKC approximately 2-fold. Kinetic studies showed that the translocation of PKC to membrane occurred before significant ingestion took place. The pharmacologic PKC inhibitor H7 inhibited IgG-dependent ingestion with ID50 of 20 microM, while the structurally related isoquinoline sulfonamide HA1004 had no effect at this concentration. Staurosporine and calphostin C, PKC inhibitors which have different mechanisms of actions than H7, also inhibited ingestion. Depletion of PKC by prolonged incubation with phorbol esters also inhibited phagocytosis, and dose-response curves showed a strong correlation between the extent of PKC depletion and the extent of inhibition of ingestion. Finally, phagosomes were isolated by sucrose density centrifugation of cells disrupted 5 min after the initiation of phagocytosis. Measurement of PKC activity and immunoreactivity in the phagosomes showed that PKC was concentrated in the phagosome membrane approximately 5-fold compared to the uninvolved plasma membrane. Together, these data suggest that PKC activation is an early, essential step in the efficient ingestion of IgG-opsonized targets by monocytes.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1992

The role of arrestin and retinoids in the regeneration pathway of rhodopsin.

Phototransduction results from a cascade of reactions that culminate in a neuronal signal. Photoisomerization of rhodopsin's chromophore, 11-cis-retinal to all-trans-retinal, leads to the formation of the activated photoproduct metarhodopsin II (Meta II). Subsequently, Meta II initiates the excitation events by activating many copies of the rod cell-specific G-proteins (Gt or transducin). To terminate the signal, the long-lived Meta II must be quenched. Deactivation of Meta II involves phosphorylation by rhodopsin kinase followed by the binding of arrestin. In order to recycle rhodopsin for phototransduction, arrestin must dissociate, and the chromophore must be replaced. In this study, we show that the reduction of the photolyzed chromophore all-trans-retinal to all-trans-retinol is essential for recycling photoactivated rhodopsin. Once this reduction has occurred, the arrestin blockade of the receptor is removed, the chromophore site becomes accessible for regeneration, and the phosphates can be hydrolyzed. If the reduction does not occur, we demonstrate that free all-trans-retinal can react with the apoprotein to form pseudo-photoproducts that are spectrally identical to the photoinduced metarhodopsin species (Meta I/II/III). The Meta II-like product, M380, interacts tightly with arrestin and kinase, however, it does not measurably interact with Gt. The persistent blockade by arrestin and the low affinity for Gt together prevent activation of the visual cascade. Therefore, any insufficiency in the reduction of all-trans-retinal to all-trans-retinol may lead to the accumulation of M380-arrestin in situ, which may effect adaptational processes.

Adenosine Triphosphate↗
PubMed1992

ProSearch: fast searching of protein sequences with regular expression patterns related to protein structure and function.

This report describes the implementation of ProSearch, a computer program that can efficiently search for motifs in protein sequences. ProSearch currently uses motifs that are contained in the PROSITE database, but user-developed patterns can easily be added to any search. ProSearch can generate a report identifying the patterns present in a given protein sequence, their locations and, if desired, a short description of the identified patterns. The program is written in AWK (a small interpreted computer language), which can run on all computer platforms commonly found in laboratories. ProSearch can search a 348-amino acid protein for 690 patterns in less than 5 s on a typical workstation.

Amino Acid Sequence↗
PubMed2004

Targeted expression of Cre recombinase to cone photoreceptors in transgenic mice.

PURPOSE: Disruption of widely expressed essential genes in mice often leads to embryonic or neonatal lethality. To circumvent this problem and dissect gene functions in the cone photoreceptors, we elected to generate cone photoreceptor specific cre transgenic mice. METHODS: Transgenic mice expressing Cre recombinase directed by the human red/green pigment (HRGP) gene promoter were generated. Candidate Cre-expressing lines were identified with RT-PCR. Cre-expressing mice were characterized with immunocytochemical assays and functional studies using a Cre activatable lacZ reporter mouse strain (R26R). Cone distribution was determined by immunohistochemistry and retinal function was measured by electroretinography (ERG) on six month old HRGP-cre mice. RESULTS: RT-PCR analysis suggested that several transgenic lines expressed cre mRNA in the retina. Beta-galactosidase staining on retinal flat mounts and sections from F1 mice derived from the HRGP-cre and R26R reporter mice suggested that two mouse strains were capable of carrying out efficient Cre mediated recombination in cone photoreceptors. Immunocytochemical staining of retinal sections demonstrated that Cre expression was localized to cone photoreceptors. Cone distribution and cone ERG analysis suggested that cone photoreceptors were normal in adult HRGP-cre mice. CONCLUSIONS: We have generated transgenic mice that efficiently express Cre recombinase in cone photoreceptor cells. The adult transgenic mice have normal cone distribution and function. They can be used in conditional knockout experiments for gene function studies in cone photoreceptors.

Animals↗
PubMed2004

Bromodeoxyuridine increases multipotency of human bone marrow-derived stem cells.

PURPOSE: Recent reports show that marrow derived mesenchymal stem cells (MeSCs) may have the ability to differentiate into diverse cell types unrelated to their phenotypical embryonic origin, including neural cells. While demonstrated "in vitro" and neonatally, efforts to demonstrate this ability in adult animal brains have had limited success. If it can be shown that human MeSC (HMeSC) can differentiate into neural cells in adult brain, it would open up the possibility that HMeSCs may be of potential therapeutic use in cell replacement therapies for neurological diseases. Here, we demonstrate that adult HMeSCs treated with 5-bromo-2-deoxyuridine (BrdU) for 3 weeks develop the capability to differentiate into neural and retinal cells when provided the appropriate lineage specific differentiation signals in vitro and in adult animals. HMeSC without BrdU treatment did not differentiate into neurons in vitro or adult animal or retinal cells in adult animal. METHODS: MeSCs isolated from adult human bone marrow were treated with BrdU (3 muM) for 3 weeks and then subjected to differentiation conditions both in vitro and in vivo. RESULTS: BrdU pretreated HMeSCs express neuronal and glial markers after co-culture with differentiated human neural stem cells and after transplantation into the adult rat brain. HMeSCs pretreated with BrdU and transforming growth factor-beta3 express a photoreceptor marker after transplantation into the adult rat vitreous. CONCLUSIONS: These results suggest that BrdU treatment may increase the multipotency of HMeSCs for possible use in autologous cell therapies for neurological and opthamological diseases.

Animals↗
PubMed2006

Mutation screening of the peropsin gene, a retinal pigment epithelium specific rhodopsin homolog, in patients with retinitis pigmentosa and allied diseases.

PURPOSE: To investigate the peropsin gene (RRH), encoding a retinal pigment epithelium homolog of the rod-expressed opsin (rhodopsin), for the presence of pathogenic mutations causing retinitis pigmentosa (RP) or other retinal degenerations. METHODS: All seven exons composing the RRH open reading frame and the immediate intron sequences were analyzed by direct nucleotide sequencing of 613 patients with forms of retinal degeneration. RESULTS: One patient with retinitis punctata albescens was a heterozygote with the missense change Cys98Tyr (TGT>TAT, c.293G>A). This change affects the homologous residue that is the target of the rhodopsin mutation Cys110Tyr, a reported cause of dominant RP. Unfortunately, none of the patient's relatives were available for a segregation analysis to determine if this change is unambiguously associated with disease. No definite pathogenic mutation was found in any of the other 612 patients who were evaluated. CONCLUSIONS: The Cys98Tyr is a possible cause of retinitis punctata albescens, although this conclusion is tentative because the change was found in only one patient. Our results indicate that the peropsin gene is not a common cause of RP or some related retinal degenerations, at least in the set of patients we analyzed.

Cysteine↗
PubMed2006

Circadian expression of clock genes in the rat eye and brain.

The light sensing system in the eye directly affects the circadian oscillator in the mammalian suprachiasmatic nucleus (SCN). To investigate this relationship in the rat, we examined the circadian expression of clock genes in the SCN and eye tissue during a 24 h day/night cycle. In the SCN, rPer1 and rPer2 mRNAs were expressed in a clear circadian rhythm like rCry1 and rCry2 mRNAs, whereas the level of BMAL1 and CLOCK mRNAs decreased during the day and increased during the night with a relatively low amplitude. It seems that the clock genes of the SCN may function in response to a master clock oscillation in the rat. In the eye, the rCry1 and rCry2 were expressed in a circadian rhythm with an increase during subjective day and a decrease during subjective night. However, the expression of Opn4 mRNA did not exhibit a clear circadian pattern, although its expression was higher in daytime than at night. This suggests that cryptochromes located in the eye, rather than melanopsin, are the major photoreceptive system for synchronizing the circadian rhythm of the SCN in the rat.

Animals↗
PubMed1991

Effects of retinal detachment on rod disc membrane assembly in cultured frog retinas.

The authors compared rod outer segment (ROS) disc membrane assembly rates in detached and attached frog retinas to determine if there was a rapid impairment of membrane assembly in response to retinal detachment. Membrane assembly was quantified in vitro by incubating retinas in medium containing Lucifer yellow, which is entrapped by nascent discs. Video microscopy was used to detect incorporation of the dye. During the first 10 hr after separation of the retina from the retinal pigment epithelium (RPE), ROS-disc membrane assembly in isolated Xenopus laevis neural retinas continued at a near normal rate, 0.81 microns/10 hr, a 13% reduction (P less than .01), compared with the 0.93 microns/10 hr observed in attached control retinas. The morphology of the OS appeared normal in most rod photoreceptors by transmission electron microscopy, although vesiculation of the most basal OS membranes was seen in a small population (25%) of rods. Approximately 90% of rod photoreceptors continued to assemble OS membranes for more than 10 hr after detachment, but by the end of 2 days, only 55% were still making new discs. The percentage of rods with normal basal OS membranes also decreased (to approximately 50%). Therefore, only 25% were assembling morphologically normal discs 2 days after detachment. In attached control regions, rod photoreceptors showed a comparatively minor response to culture conditions; assembly of morphologically normal discs continued for 2 days in about 85% and ceased in only 10%. These results indicate that the effects on disc membrane assembly of disrupting photoreceptor-RPE interaction in vitro initially are slight but become progressively severe with time.

Animals↗
PubMed1990

Holistic approaches to receptor and channel structure and dynamics.

A detailed three-dimensional structure of a molecule is a necessary, but not sufficient prerequisite to understanding its behaviour. Crystal structures are lacking for protein receptors and ion channels. However, the amino acid sequences for representatives of the most important superfamilies of neuroactive proteins (ligand-gated ion channels, voltage-gated ion channels and G-protein receptors) are known. To address the problem of three-dimensional structure and, subsequently, the dynamic properties of these molecules, a holistic approach was applied to the nicotinic acetylcholine receptor (nAChR), the sodium channel of nerves (NaC) and rhodopsin (Rh) to build 'working' structures. For nAChR and Rh, the chains are placed within an envelope derived from image analysis of electron micrographs. Physical organic principles suggest how to juxtapose binding groups as well as structures for ion channels, leading to the identification of the most common feature of channels, the positively charged 'S4' segment and the ever-present amphiphilic segment of nAChR proteins, which has homologous counterparts in the glutamate receptor and glutamate-binding proteins. The cyclic GMP-gated NaC (cGMP-NaC) is related to the eel and rat neuronal NaC, and has a novel positively charged control sequence.

Amino Acid Sequence↗