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R Sullivan

Publications and source records attributed to R Sullivan.

At least 37 records · Page 2Linked to original sources
PubMed2000

Calmodulin regulates the disassembly of cortical F-actin in mast cells but is not required for secretion.

Secretion is dependent on a rise in cytosolic Ca(2+)concentration and is associated with dramatic changes in actin organization. The actin cortex may act as a barrier between secretory vesicles and plasma membrane. Thus, disassembly of this cortex should precede late steps of exocytosis. Here we investigate regulation of both the actin cytoskeleton and secretion by calmodulin. Ca(2+), together with ATP, induces cortical F-actin disassembly in permeabilized rat peritoneal mast cells. This effect is strongly inhibited by removing endogenous calmodulin (using calmodulin inhibitory peptides), and increased by exogenous calmodulin. Neither treatment, however, affects secretion. Low concentrations ( approximately 1 microM) of a specific inhibitor of myosin light chain kinase, ML-7, prevent F-actin disassembly, but not secretion. In contrast, a myosin inhibitor affecting both conventional and unconventional myosins, BDM, decreases cortical disassembly as well as secretion. Observations of fluorescein-calmodulin, introduced into permeabilized cells, confirmed a strong (Ca(2+)-independent) association of calmodulin with the actin cortex. In addition, fluorescein-calmodulin enters the nuclei in a Ca(2+)-dependent manner. In conclusion, calmodulin promotes myosin II-based contraction of the membrane cytoskeleton, which is a prerequisite for its disassembly. The late steps of exocytosis, however, require neither calmodulin nor cortical F-actin disassembly, but may be modulated by unconventional myosin(s).

Actins↗
PubMed2000

Exobiology.

Explore the source record for details and available documents.

Exobiology↗
PubMed2000

Coexpression of full-length gamma-aminobutyric acid(B) (GABA(B)) receptors with truncated receptors and metabotropic glutamate receptor 4 supports the GABA(B) heterodimer as the functional receptor.

Direct evidence is lacking to show whether the gamma-aminobutyric acid (GABA)(B) gb1-gb2 heterodimer is the signaling form of the receptor. In this study, we tested whether gb1a or gb2 subunits when coexpressed with truncated receptors or metabotropic glutamate receptor mGluR4 could form functional GABA receptors. Coexpression of the ligand binding N-terminal domain of gb1a or the C-terminal portion of gb1a composing the seven-transmembrane segments and intracellular loops with gb2 could not reconstitute functional receptors. We next examined whether mGluR4, which forms homodimers and is structurally related to GABA(B), could act as a surrogate coreceptor for gb1 or gb2. The coexpression of mGluR4 and gb1a led to the expression of gb1a monomers on cell surface membranes as determined by immunoblot analysis and flow cytometry. However, mGluR4-gb1a heterodimers were not formed, and membrane-expressed gb1a monomers were not functionally coupled to adenylyl cyclase in human embryonic kidney 293 cells or activated inwardly rectifying potassium (Kir) channels in Xenopus oocytes. Similarly, the coexpression of mGluR4 and gb2 led to nonfunctional GABA receptors. GABA-activated distal signaling events resulted only after the coexpression and heterodimerization of gb1 and gb2. Taken together with the truncated receptor studies, the data suggest that a high degree of structural specificity is required to form the functional GABA(B) receptor that is a gb1-gb2 heterodimer.

Amino Acid Sequence↗
PubMed1999

Rho controls cortical F-actin disassembly in addition to, but independently of, secretion in mast cells.

Localized disassembly of cortical F-actin has long been considered necessary for facilitation of exocytosis. Exposure of permeabilized mast cells to calcium/ATP induces cortical F-actin disassembly (calmodulin-dependent) and secretion (calmodulin-independent). The delay in the onset of secretion is characteristic for the calcium/ATP response and is abolished by GTP. Here we report that a constitutively active mutant of Rho (V14RhoA) enhanced both secretion and cortical F-actin disassembly. In addition, V14RhoA mimicked GTP by abolishing the delay in secretion. Inhibition of Rho by C3 transferase prevented both secretion ( approximately 80%) and F-actin disassembly (approximately 20%). Thus, both Rho GTPase and calcium/calmodulin contribute to the control of cortical F-actin disassembly. Stabilization of actin filaments by high concentrations of phalloidin or by a calmodulin-inhibitory peptide (based on the calmodulin-binding domain of myosin light chain kinase) did not affect the extent of secretion or the secretion-enhancing effects of V14RhoA. These results further support the existence of divergent, Rho-dependent, pathways regulating actin and exocytosis. Furthermore, compound Y-27632, a specific inhibitor of Rho-associated protein kinase (p160(ROCK)), attenuated the Rho-induced loss of cortical F-actin without affecting secretion. A model is presented in which Rho regulates secretion and cortical F-actin in a manner dependent on and/or synergistic with calcium.

Actins↗
PubMed1999

Identification of a GABAB receptor subunit, gb2, required for functional GABAB receptor activity.

G protein-coupled receptors are commonly thought to bind their cognate ligands and elicit functional responses primarily as monomeric receptors. In studying the recombinant gamma-aminobutyric acid, type B (GABAB) receptor (gb1a) and a GABAB-like orphan receptor (gb2), we observed that both receptors are functionally inactive when expressed individually in multiple heterologous systems. Characterization of the tissue distribution of each of the receptors by in situ hybridization histochemistry in rat brain revealed co-localization of gb1 and gb2 transcripts in many brain regions, suggesting the hypothesis that gb1 and gb2 may interact in vivo. In three established functional systems (inwardly rectifying K+ channel currents in Xenopus oocytes, melanophore pigment aggregation, and direct cAMP measurements in HEK-293 cells), GABA mediated a functional response in cells coexpressing gb1a and gb2 but not in cells expressing either receptor individually. This GABA activity could be blocked with the GABAB receptor antagonist CGP71872. In COS-7 cells coexpressing gb1a and gb2 receptors, co-immunoprecipitation of gb1a and gb2 receptors was demonstrated, indicating that gb1a and gb2 act as subunits in the formation of a functional GABAB receptor.

Amino Acid Sequence↗
PubMed1999

Hamster sperm antigen P26h is a phosphatidylinositol-anchored protein.

We have previously identified a hamster sperm protein, P26h, proposed to be involved in the interaction between spermatozoa and the egg's zona pellucida. In this study we investigated the mechanism of P26h accumulation on hamster spermatozoa during epididymal maturation. Immunocytochemical studies showed an accumulation of P26h on the acrosomal cap of hamster spermatozoa during epididymal transit. To document the anchoring mechanism of P26h, cauda epididymal spermatozoa were exposed to different treatments. High-salt buffered solutions were unable to remove P26h from the surface of intact spermatozoa. P26h was released in a dose-dependent manner when live spermatozoa were treated with a solution of phospholipase C specific to phosphatidylinositol. In contrast, the P26h remained associated to the sperm surface following treatment with trypsin. To document the transfer mechanisms of P26h on the maturing spermatozoa, prostasomes were isolated from the epididymal fluid and subjected to immunodetection. Western blots and immunogold studies showed that P26h was associated to epididymal prostasomes. Phospholipase C treatment performed on epididymal prostasomes, indicated that P26h also is anchored to these vesicles via a phosphatidylinositol. These results suggest that epididymal sperm maturation involves a cell to cell transfer of a phosphaditylinositol-anchored protein and that prostasomes may be implicated in this process.

Alcohol Oxidoreductases↗
PubMed1999

Identification of capacitation-associated phosphoproteins in porcine sperm electroporated with ATP-gamma-(32)P.

Our objectives were to incorporate ATP-gamma-(32)P into boar sperm to radiolabel endogenous phosphoproteins and compare phosphorylation patterns from sperm incubated in capacitating (CM) and non-capacitating conditions (NCM). Sperm were electroporated (1000 V/cm, 125 microF/cm, 65 Omega/cm, 0.3 msec) with ATP-gamma-(32)P which moderately decreased sperm viability (P < 0.01), but did not affect motility (P = 0.34) or the appearance of spontaneous acrosome reactions (P = 0.49). Sperm incubated in CM for 3 hr underwent capacitation, determined by the ability to undergo ionophore-induced acrosome reactions (P </= 0.05). Furthermore, more sperm in CM than in NCM exhibited chlortetracycline (CTC) pattern B (capacitated) fluorescence (P </= 0.01). SDS-PAGE, autoradiography and phosphoimagery of extracted, (32)P-labeled sperm proteins revealed a subset of phosphoproteins (Mr 28,000-60,000) from cells incubated in CM, whereas only two phosphorylated proteins were evident from sperm in NCM (44 and 57 kDa). The appearance of phosphoproteins increased concomitant with capacitation (P </= 0.05). In NCM, the 44 kDa protein was unaffected by time (P > 0.05) and the 57 kDa phosphoprotein increased after capacitation (P </= 0.05). Computer-assisted analysis revealed that the percentage of motile sperm in either medium decreased with time, and CM only transiently maintained motility over NCM (P >/= 0.02). ATP-gamma-(32)P can, therefore, be incorporated into porcine sperm to radiolabel endogenous phosphoproteins, and the different profiles from sperm incubated in NCM versus CM suggest that capacitation is mediated by signaling events involving protein phosphorylation.

Adenosine Triphosphate↗
PubMed1999

Toward improved management of NIDDM: A randomized, controlled, pilot intervention using a lowfat, vegetarian diet.

OBJECTIVE: To investigate whether glycemic and lipid control in patients with non-insulin-dependent diabetes (NIDDM) can be significantly improved using a low-fat, vegetarian (vegan) diet in the absence of recommendations regarding exercise or other lifestyle changes. METHODS: Eleven subjects with NIDDM recruited from the Georgetown University Medical Center or the local community were randomly assigned to a low-fat vegan diet (seven subjects) or a conventional low-fat diet (four subjects). Two additional subjects assigned to the control group failed to complete the study. The diets were not designed to be isocaloric. Fasting serum glucose, body weight, medication use, and blood pressure were assessed at baseline and biweekly thereafter for 12 weeks. Serum lipids, glycosylated hemoglobin, urinary albumin, and dietary macronutrients were assessed at baseline and 12 weeks. RESULTS: Although the sample was intentionally small in accordance with the pilot study design, the 28% mean reduction in fasting serum glucose of the experimental group, from 10.7 to 7.75 mmol/L (195 to 141 mg/dl), was significantly greater than the 12% decrease, from 9.86 to 8.64 mmol/L (179 to 157 mg/dl), for the control group (P < 0.05). The mean weight loss was 7.2 kg in the experimental group, compared to 3. 8 kg for the control group (P < 0.005). Of six experimental group subjects on oral hypoglycemic agents, medication use was discontinued in one and reduced in three. Insulin was reduced in both experimental group patients on insulin. No patient in the control group reduced medication use. Differences between the diet groups in the reductions of serum cholesterol and 24-h microalbuminuria did not reach statistical significance; however, high-density lipoprotein concentration fell more sharply (0.20 mmol/L) in the experimental group than in the control group (0.02 mmol/L) (P < 0.05). CONCLUSION: The use of a low-fat, vegetarian diet in patients with NIDDM was associated with significant reductions in fasting serum glucose concentration and body weight in the absence of recommendations for exercise. A larger study is needed for confirmation.

Adult↗
PubMed1999

Synthesis of the nanomolar photoaffinity GABA(B) receptor ligand CGP 71872 reveals diversity in the tissue distribution of GABA(B) receptor forms.

A radioiodinated probe, [125I]-CGP 71872, containing an azido group that can be photoactivated, was synthesized and used to characterize GABA(B) receptors. Photoaffinity labeling experiments using crude membranes prepared from rat brain revealed two predominant ligand binding species at approximately 130 and approximately 100 kDa believed to represent the long (GABA(B)R1a) and short (GABA(B)R1b) forms of the receptor. Indeed, these ligand binding proteins were immunoprecipitated using a GABA(B) receptor-specific antibody confirming the receptor specificity of the photoaffinity probe. Most convincingly, [125I]-CGP 71872 binding was competitively inhibited in a dose-dependent manner by cold CGP 71872, GABA, saclofen, (-)-baclofen, (+)-baclofen and (L)-glutamic acid with a rank order and stereospecificity characteristic of the GABA(B) receptor. Photoaffinity labeling experiments revealed that the recombinant GABA(B)R2 receptor does not bind [125I]-CGP 71872, providing surprising and direct evidence that CGP 71872 is a GABA(B)R1 selective antagonist. Photoaffinity labeling experiments using rat tissues showed that both GABA(B)R1a and GABA(B)R1b are co-expressed in the brain, spinal cord, stomach and testis, but only the short GABA(B)R1b receptor form was detected in kidney and liver whereas the long GABA(B)R1a form was selectively expressed in the adrenal gland, pituitary, spleen and prostate. We report herein the synthesis and biochemical characterization of the nanomolar affinity [125I]-CGP 71872 and CGP 71872 GABA(B)R1 ligands, and differential tissue expression of the long GABA(B)R1a and short GABA(B)R1b receptor forms in rat and dog.

Affinity Labels↗
PubMed1999

Hamster sperm protein, p26h: a member of the short-chain dehydrogenase/reductase superfamily.

For successful fertilization to occur, mammalian spermatozoa must undergo a series of modifications in order to reach and penetrate the oocyte. Some of these modifications occur during passage through the epididymis, the site where spermatozoa acquire their fertilizing ability. We have previously described hamster sperm protein, P26h, which is acquired by spermatozoa during epididymal transit, and have proposed that this protein is involved in sperm-egg binding. In the present study, we report the cloning and characterization of the full-length cDNA encoding hamster P26h. A database search using the predicted hamster P26h amino acid sequence revealed 85% identity with mouse AP27 protein and porcine carbonyl reductase, members of the short-chain dehydrogenase/reductase (SDR) family of proteins. Northern blot analysis revealed a major P26h 1-kilobase transcript in the testis. No signal was detected in other somatic tissues of the hamster. In situ hybridization experiments revealed that the P26h gene was predominantly transcribed in seminiferous tubules of the testis and at a lower level in the corpus epididymidis. The identity of the cloned P26h was confirmed by immunoprecipitating in vitro-translated P26h using polyclonal antiserum raised against purified hamster sperm P26h. Taken together, these results identify P26h as a new member of the SDR family of proteins involved in the processes of mammalian gamete interactions.

Alcohol Oxidoreductases↗
PubMed1999

P34H sperm protein is preferentially expressed by the human corpus epididymidis.

During epididymal transit, mammalian spermatozoa acquire new surface proteins that are necessary for gamete interaction. We have previously described a 34-kDa human epididymal sperm protein, P34H, that has been shown to be involved in sperm-zona pellucida interaction. In the present study, we report the cloning and characterization of the full-length complementary DNA encoding human P34H. The predicted amino acid sequence revealed 65% identity with P26h, the hamster counterpart of the P34H. The deduced P34H amino acid sequence revealed a 71% similarity with a pig lung tetrameric carbonyl reductase, a member of the short chain dehydrogenase/ reductase family proteins. Northern blot analysis revealed that P34H messenger RNA (mRNA) was highly expressed in the human epididymis, principally in the corpus region. A single 912-bp P34H transcript was detected. In situ hybridization experiments showed that the P34H mRNA was predominantly expressed in the proximal and distal sections of the corpus epididymidis. The staining was restricted to the principal cells of the epididymal epithelium. The localization of P34H mRNA was in agreement with the appearance of P34H protein along the male reproductive tract. Western blot analysis revealed that recombinant P34H expressed by a yeast expression system, is antigenically related to the native P34H sperm protein. Based on its pattern of expression and its function in one of the key steps leading to fertilization, P34H can be considered as a marker of epididymal sperm maturation in humans.

Adult↗
PubMed1998

Heart malformations in transgenic mice exhibiting dominant negative inhibition of gap junctional communication in neural crest cells.

Transgenic mice were generated expressing an alpha1 connexin/beta-galactosidase fusion protein previously shown to exert dominant negative effects on gap junctional communication. RNase protection analysis and assays for beta-galactosidase enzymatic activity showed that the transgene RNA and protein are expressed in the embryo and adult tissues. In situ hybridization analysis revealed that in the embryo, expression was predominantly restricted to neural crest cells and their progenitors in the dorsal neural tube, regions where the endogenous alpha1 connexin gene is also expressed. Dye-coupling analysis indicated that gap junctional communication was inhibited in the cardiac neural crest cells. All of the transgenic lines were homozygote inviable, dying neonatally and exhibiting heart malformations involving the right ventricular outflow tract-the same region affected in the alpha1 connexin knockout mice. As in the knockout mice, the conotruncal heart malformations were accompanied by outflow tract obstruction. Histological analysis showed that this was associated with abnormalities in the differentiation of the conotruncal myocardium. These results suggest that the precise level of gap junctional communication in cardiac neural crest cells is of critical importance in right ventricular outflow tract morphogenesis. Consistent with this possibility is the fact that cardiac crest cells from the alpha1 connexin knockout mice also exhibited a greatly reduced level of gap junctional communication. These studies show the efficacy of a dominant negative approach for manipulating gap junctional communication in the mouse embryo and demonstrate that targeted expression of this fusion protein can be a powerful tool for examining the role of gap junctions in mammalian development.

Animals↗
PubMed1998

Orbital lymphosarcoma associated with reticuloendotheliosis virus in a peafowl.

Lymphosarcoma associated with infection by avian reticuloendotheliosis virus was diagnosed in an Indian peafowl with exophthalmia and exposure keratitis. Exenteration of the orbit was complicated by a profound oculocardiac reflex and extensive hemorrhage during surgery. Orbital bleeding was controlled by direct pressure, electrocautery, topical administration of bovine thrombin, and application of sterile gelatin sponges and oxidized regenerated cellulose. A blood transfusion was also performed. In addition to describing methods of handling intraoperative complications of orbital exenteration in birds, to the authors' knowledge, this is the first report to describe an association of reticuloendotheliosis virus, which more commonly affects poultry, with lymphosarcoma in an Indian peafowl.

Animals↗
PubMed1998

Evidence for a subsurface ocean on Europa.

Ground-based spectroscopy of Jupiter's moon Europa, combined with gravity data, suggests that the satellite has an icy crust roughly 150 km thick and a rocky interior. In addition, images obtained by the Voyager spacecraft revealed that Europa's surface is crossed by numerous intersecting ridges and dark bands (called lineae) and is sparsely cratered, indicating that the terrain is probably significantly younger than that of Ganymede and Callisto. It has been suggested that Europa's thin outer ice shell might be separated from the moon's silicate interior by a liquid water layer, delayed or prevented from freezing by tidal heating; in this model, the lineae could be explained by repetitive tidal deformation of the outer ice shell. However, observational confirmation of a subsurface ocean was largely frustrated by the low resolution (>2 km per pixel) of the Voyager images. Here we present high-resolution (54 m per pixel) Galileo spacecraft images of Europa, in which we find evidence for mobile 'icebergs'. The detailed morphology of the terrain strongly supports the presence of liquid water at shallow depths below the surface, either today or at some time in the past. Moreover, lower-resolution observations of much larger regions suggest that the phenomena reported here are widespread.

Extraterrestrial Environment↗
PubMed1998

Evidence for non-synchronous rotation of Europa. Galileo Imaging Team.

Non-synchronous rotation of Europa was predicted on theoretical grounds, by considering the orbitally averaged torque exerted by Jupiter on the satellite's tidal bulges. If Europa's orbit were circular, or the satellite were comprised of a frictionless fluid without tidal dissipation, this torque would average to zero. However, Europa has a small forced eccentricity e approximately 0.01 , generated by its dynamical interaction with Io and Ganymede, which should cause the equilibrium spin rate of the satellite to be slightly faster than synchronous. Recent gravity data suggest that there may be a permanent asymmetry in Europa's interior mass distribution which is large enough to offset the tidal torque; hence, if non-synchronous rotation is observed, the surface is probably decoupled from the interior by a subsurface layer of liquid or ductile ice. Non-synchronous rotation was invoked to explain Europa's global system of lineaments and an equatorial region of rifting seen in Voyager images. Here we report an analysis of the orientation and distribution of these surface features, based on initial observations made by the Galileo spacecraft. We find evidence that Europa spins faster than the synchronous rate (or did so in the past), consistent with the possibility of a global subsurface ocean.

Jupiter↗
PubMed1998

Episodic plate separation and fracture infill on the surface of Europa. Galileo Imaging Team.

Images obtained by the Voyager spacecraft revealed dark, wedge-shaped bands on Europa that were interpreted as evidence that surface plates, 50-100 km across, moved and rotated relative to each other. This implied that they may be mechanically decoupled from the interior by a layer of warm ice or liquid water. Here we report similar features seen in higher resolution images (420 metres per pixel) obtained by the Galileo spacecraft that reveal new details of wedge-band formation. In particular, the interior of one dark band shows bilateral symmetry of parallel lineaments and pit complexes which indicates that plate separation occurred in discrete episodes from a central axis. The images also show that this style of tectonic activity involved plates < 10 km across. Although this tectonic style superficially resembles aspects of similar activity on Earth, such as sea-floor spreading and the formation of ice leads in polar seas, there are significant differences in the underlying physical mechanisms: the wedge-shaped bands on Europa most probably formed when lower material (ice or water) rose to fill the fractures that widened in response to regional surface stresses.

Geological Phenomena↗
PubMed1998

Isolation of antiSLIP1-reactive boar sperm P68/62 and its binding to mammalian zona pellucida.

Single-step purification of boar sperm P68/62 that is cross-reactive with a polyclonal antibody against sulfolipidimmobilizing protein 1 (SLIP1) was achieved by chromatofocusing. This method is useful for obtaining P68/62 in quantity. The two proteins, P68 and P62, were antigenically related, since the antibody generated specifically against the 68-kDa band reacted with both the 68- and 62-kDa bands. Like rat testis SLIP1, purified boar sperm P68/62 bound to sulfogalactosylglycerolipid (SGG) and inhibited sperm-egg binding in a dose-dependent manner when added exogenously to sperm-egg coincubates. This inhibitory effect occurred at the level of the zona pellucida (ZP), and further studies showed that biotinylated boar sperm P68/62 bound to the ZP of unfertilized mouse eggs. Furthermore, biotinylated boar sperm P68/62 bound to isolated ZP of unfertilized eggs from other species, including pig, rat, cat, dog, and human, as well as to ZP of intact fertilized mouse eggs and preimplantation embryos of various developmental stages, although the degree of its binding to the ZP of intact eight-cell embryos, morulae, and blastocysts was much lower than that of fertilized eggs and two-cell embryos. These results suggest that P68/62 of capacitated sperm must act together with other sperm surface proteins/molecules that regulate zona binding specificity within homologous species and in unfertilized eggs. Together with our previous findings, we suggest that rather than being a true ZP receptor, sperm P68/62 may be involved in the initial step of sperm-ZP binding that is adhesive in nature.

Animals↗