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Y K Ho

Publications and source records attributed to Y K Ho.

At least 37 records · Page 2Linked to original sources
PubMed1996

The activation of murine macrophages and natural killer cells by the partially thiolated double stranded RNA poly(I)-mercapto poly(C).

Partially thiolated analogs of the biological response modifier poly I.poly C (pI.pC) were synthesized. Each of these analogs (pI.MPC) contained a partially thiolated polycytidylate (MPC) strand containing either 1.2%, 4.6%, or 17% 5-mercaptocytosine (%SH) randomly introduced throughout the polynucleotide. The ability of these double stranded RNAs (dsRNAs) to activate murine peritoneal macrophages in vitro and augment natural killer (NK) cell activity in mice following intraperitoneal (i.p.) administration was determined. Macrophages were treated in vitro for 24 hours with pI.pC, or pI.MPC, washed, and then overlayed with exponentially growing L1210 leukemia cells at an effector to target (E:T) ratio of 25:1. The cytostatic effect of the macrophages on the L1210 cells was determined by 3H.thymidine pulse labelling. The rank order of potency for macrophage activation was determined to be: pI.pC>pI.MPC(1.2% SH)>pI.MPC(4.6% SH)pI.MPC(17% SH). Twenty hours following i.p. administration of 5 mg/kg of each pI.MPC analog, splenic NK cell activity was assessed in a standard 51Cr release assay using the murine tumor target cell line YAC- 1. The rank order of potency observed for NK cell activation was determined to be; pI.pCpI MPC(1.2% SH)>pI.MPC(4.6% SH)>pI.MPC(17% SH). These dsRNAs activated NK cells in a dose dependent manner. The efficacy and time course for NK cell activation following i.p. administration of pI.MPC (1.2% SH) at a dose of 10 mg/kg was directly compared to an equivalent 10 mg/kg i.p. dose of pI.pC. NK cell activation took place within three hours following treatment with pI-pC whereas the onset of NK cell activation by pI.MPC (1.2%) occurred between 8 and 20 hours post treatment. NK cell activity steadily declined from 24 to 50 hours post treatment at which time the NK activity in both treatment groups was similar. There was a significant correlation between the immunostimulatory potency of these dsRNAs and their experimentally determined melting temperatures (r2 = 0.88) and percent hyperchromicity upon thermal denaturation (r2 = 0.99). At the lower %SH, pI.MPC retains most of the immunostimulatory activities of p1.pC and may serve as a useful and potent biological response modifier.

Animals↗
PubMed1995

Hairpin orientation of sterol regulatory element-binding protein-2 in cell membranes as determined by protease protection.

Sterol regulatory element-binding proteins (SREBP-1 and SREBP-2) are proteins of approximately 1150 amino acids each that are attached to membranes of the endoplasmic reticulum (ER). In sterol-depleted cells, a protease releases an NH2-terminal fragment of approximately 500 amino acids that contains a basic helix-loop-helix leucine zipper motif. This fragment enters the nucleus and stimulates transcription of genes encoding the low density lipoprotein receptor and enzymes of cholesterol biosynthesis. Prior evidence indicates that the SREBPs are attached to membranes by virtue of an 80-residue segment located approximately 80 amino acids to the COOH-terminal side of the leucine zipper. This segment contains two long hydrophobic sequences separated by a short hydrophilic sequence of approximately 30 amino acids. We have proposed a hairpin model in which the two hydrophobic sequences span the membrane, separated by the short hydrophilic sequence which projects into the lumen of the ER (the "lumenal loop"). The model predicts that the NH2- and COOH-terminal segments face the cytosol. To test this model, we constructed a cDNA encoding human SREBP-2 with epitope tags at the NH2 terminus and in the lumenal loop. The COOH-terminal region was visualized with a newly developed monoclonal antibody against this region. Sealed membrane vesicles were isolated from cells expressing the epitope-tagged version of SREBP-2. Trypsin treatment of these vesicles destroyed the NH2- and COOH-terminal segments and reduced the lumenal epitope to a size consistent with protection of the lumenal sequence plus the two membrane-spanning segments. The lumenal epitope tag contained two potential sites for N-linked glycosylation. The size of the trypsin-protected fragment was reduced by treatment with N-Glycanase and endoglycosidase H, indicating that this segment was located in the lumen of the ER where it was glycosylated. These data provide strong support for the hairpin model.

Amino Acid Sequence↗
PubMed1995

Deficient geranylgeranylation of Ram/Rab27 in choroideremia.

Choroideremia, an X-linked form of retinal degeneration, results from defects in the Rab escort protein-1 (REP-1) gene. REP-1 and REP-2 assist in the attachment of geranylgeranyl groups to Rab GTPases, a modification essential for their action as molecular switches regulating intracellular vesicular transport. If Rabs that depend preferentially on REP-1 for prenylation exist, they will accumulate unprenylated in choroideremia cells. Using recombinant Rab geranylgeranyl transferase and REPs to label unprenylated cytosolic proteins, we identified one unprenylated protein in choroideremia lymphoblasts that was prenylated in vitro more efficiently by REP-1 than by REP-2. This protein was purified and identified as Ram (renamed Rab27), a previously cloned Rab of unknown function. Immunohistochemistry of rat retina showed that Ram/Rab27 is expressed in the pigment epithelium and choriocapillaris, the two retinal cell layers that degenerate earliest in choroideremia. These results raise the possibility that the retinal degeneration in choroideremia results from the deficient geranylgeranylation of Ram/Rab27 or a closely related protein.

Adaptor Proteins, Signal Transducing↗
PubMed1995

Three different rearrangements in a single intron truncate sterol regulatory element binding protein-2 and produce sterol-resistant phenotype in three cell lines. Role of introns in protein evolution.

The cholesterol analogue 25-hydroxycholesterol kills animal cells by blocking the proteolytic activation of two sterol-regulated transcription factors designated sterol regulatory element binding protein-1 and -2 (SREBP-1 and SREBP-2). These proteins, each approximately 1150 amino acids in length, are embedded in the membranes of the nucleus and endoplasmic reticulum by virtue of hydrophobic COOH-terminal segments. In cholesterol-depleted cells the proteins are cleaved to release soluble NH2-terminal fragments of approximately 480 amino acids that enter the nucleus and activate genes encoding the low density lipoprotein receptor and enzymes of cholesterol synthesis. 25-Hydroxycholesterol blocks this cleavage, and cells die of cholesterol deprivation. We previously described a mutant 25-hydroxycholesterol-resistant hamster cell line (SRD-1 cells) in which the SREBP-2 gene had undergone a recombination between the intron following codon 460 and an intron in an unrelated gene. The SREBP-2 sequence terminated at residue 460, eliminating the membrane attachment domain and producing a constitutively active factor that no longer required proteolysis and thus was not inhibited by 25-hydroxycholesterol. Here, we report that two additional sterol-resistant cell lines (SRD-2 and SRD-3) have also undergone genomic rearrangements in the intron following codon 460 of the SREBP-2 gene. Although the molecular rearrangements differ in the three mutant lines, each leads to the production of a constitutively active transcription factor whose SREBP-2 sequence terminates at residue 460. These findings provide a dramatic illustration of the advantage that introns provide in allowing proteins to gain new functions in response to new environmental challenges.

Amino Acid Sequence↗
PubMed1995

Reduced light-dependent phosphorylation of an analog visual pigment containing 9-demethylretinal as its chromophore.

9-Demethyl rhodopsin (9dR), an analog of vertebrate rhodopsin, consists of opsin and a covalently attached chromophore of 11-cis 9-demethylretinal. Electrophysiological evidence that photoactivated 9dR (9dR*) undergoes abnormally slow deactivation in salamander rods (Corson, D. W., Cornwall, M. C., and Pepperberg, D. R. (1994) Visual Neurosci. 11, 91-98) raises the possibility that opsin phosphorylation, a reaction involved in visual pigment deactivation, operates abnormally on 9dR*. This possibility was tested by measuring the light-dependent phosphorylation of 9dR in preparations obtained from bovine rod outer segments. Outer segment membranes containing 9dR or regenerated rhodopsin were flash-illuminated in the presence of [gamma-32P]ATP and rhodopsin kinase, further incubated in darkness, and then analyzed for opsin-bound [32P]Pi. [32P]Pi incorporation by 9dR* increased with both incubation period and bleaching extent but, under all conditions tested, was less than that measured in rhodopsin controls. Results obtained with 30-s incubation periods indicated that the maximal initial rate of incorporation by 9dR* is about 25% of that by photoactivated rhodopsin. The results imply that the low incorporation of Pi by 9dR* results from a reduced rate of phosphorylation by rhodopsin kinase and are consistent with the prolonged lifetime of 9dR* determined electrophysiologically.

Animals↗
PubMed1995

Modulation of the serotonin-activated K+ channel by G protein subunits and nucleotides in rat hippocampal neurons.

In hippocampal neurons, 5-hydroxytryptamine (5-HT) activates an inwardly rectifying K+ current via G protein. We identified the K+ channel activated by 5-HT (K5-HT channel) and studied the effects of G protein subunits and nucleotides on the K+ channel kinetics in adult rat hippocampal neurons. In inside-out patches with 10 microM 5-HT in the pipette, application of GTP (100 microM) to the cytoplasmic side of the membrane activated an inwardly rectifying K+ channel with a slope conductance of 36 +/- 1 pS (symmetrical 140 mM K+) at -60 mV and a mean open time of 1.1 +/- 0.1 msec (n = 5). Transducin beta gamma activated the K5-HT channels and this was reversed by alpha-GDP. Whether the K5-HT channel was activated endogenously (GTP, GTP gamma S) or exogenously (beta gamma), the presence of 1 mM ATP resulted in a approximately 4-fold increase in channel activity due in large part to the prolongation of the open time duration. These effects of ATP were irreversible and not mimicked by AMPPMP, suggesting that phosphorylation might be involved. However, inhibitors of protein kinases A and C (H-7, staurosporine) and tyrosine kinase (tyrphostin 25) failed to block the effect of ATP. These results show that G beta gamma activates the G protein-gated K+ channel in hippocampal neurons, and that ATP modifies the gating kinetics of the channel, resulting in increased open probability via as yet unknown pathways.

Adenosine Triphosphate↗
PubMed1994

Regulation of the very low density lipoprotein receptor by thyroid hormone in rat skeletal muscle.

A new member of the low density lipoprotein receptor gene family that binds and internalizes very low density lipoprotein (VLDL) particles was previously cloned and characterized from the rabbit and human. The physiological role of this putative VLDL receptor is not known, but its tissue distribution and ligand specificity suggest a possible role in the delivery of triglycerides to peripheral tissue. To learn more about the potential function of this receptor, we measured the changes in VLDL receptor mRNA and protein in various tissues following dietary or hormonal manipulation of rats. No significant changes in the VLDL receptor mRNA or protein were seen after a 48-h fast and subsequent to refeeding. A striking change in receptor mRNA and protein was observed in skeletal muscle of hypothyroid and hyperthyroid rats. In hypothyroid rats, the amount of immunodetectable VLDL receptor was reduced by 80%, while in the hyperthyroid animals it was increased by 300%. These maneuvers did not affect VLDL receptor mRNA or protein levels in adipose tissue or heart. The changes in VLDL receptor mRNA in muscle were opposite to those observed with lipoprotein lipase. These studies suggest that the VLDL receptor plays a role in a metabolic process in muscle that is regulated by thyroid hormone.

Amino Acid Sequence↗
PubMed1994

Assignment of the membrane attachment, DNA binding, and transcriptional activation domains of sterol regulatory element-binding protein-1 (SREBP-1).

Transcription of the low density lipoprotein receptor gene and other sterol-regulated genes is stimulated by sterol regulatory element-binding protein-1 (SREBP-1), a basic-helix-loop-helix-leucine zipper (bHLH-ZIP) transcription factor. Human SREBP-1 is synthesized as an 1147-amino acid precursor that is attached intrinsically to membranes of the nuclear envelope and endoplasmic reticulum. In sterol-depleted cells the precursor is cleaved to generate an NH2-terminal fragment that enters the nucleus and activates transcription by binding to sterol regulatory element-1 (SRE-1). Sterols prevent transcriptional activation by blocking the proteolytic cleavage. In the current studies, performed with hamster SREBP-1, we used mutational analysis to localize the transcriptional activation domain to an acidic NH2-terminal sequence. Deletion of this sequence converted SREBP-1 from an activator to an inhibitor of transcription. DNA binding was assigned to the basic region of the bHLH-ZIP domain. Binding was abolished by substitution of 3 amino acids that were previously implicated in DNA binding by Max, another bHLH-ZIP protein. The membrane attachment domain was localized to two hydrophobic regions at residues 477-497 and 536-556. Truncation of SREBP-1 prior to these regions gave rise to an NH2-terminal fragment that was soluble and entered the nucleus. This fragment was more than 30-fold more active than full-length SREBP-1 in stimulating transcription of an SRE-1 containing reporter gene in transfected cells. Deletion of the hydrophobic sequences (delta 476-556) yielded a protein that appeared cytosolic by immunofluorescence microscopy but failed to enter the nucleus readily, apparently because of inhibition by sequences in the remaining COOH-terminal domain. This study provides a picture of the domain structure of SREBP-1 and further elucidates the mechanism by which it adjusts gene transcription to maintain cholesterol homeostasis in animal cells.

Amino Acid Sequence↗
PubMed1994

Muscarinic K+ channels are activated by beta gamma subunits and inhibited by the GDP-bound form of alpha subunit of transducin.

It was reported that transducin beta gamma subunits (T beta gamma) inhibited cardiac muscarinic K+ (KACh) channels activated through GK. This result has been used to support the proposal that GK activates this channel through its alpha subunits. Here, we have reexamined the effects of transducin subunits on KACh channels. T beta gamma applied to the internal side of the inside-out patch membrane activated KACh channels in a concentration-dependent manner (EC50 approximately 1 microM). In contrast, the GDP-bound form of transducin alpha subunits (50 nM-3 microM), but not T beta gamma (1 nM-1 microM), irreversibly inhibited KACh channel openings induced either by GTP plus acetylcholine or by GTP gamma S (guanosine 5'-3-O-(thio)triphosphate). This inhibition was antagonized by exogenously applied T beta gamma (600 nM-3 microM) or brain G protein beta gamma subunits (10-30 nM). These data, opposite to the previous report, indicate that GK activates KACh channels through its beta gamma subunits.

Animals↗
PubMed1993

Mutational analysis of alpha-subunit of protein farnesyltransferase. Evidence for a catalytic role.

Protein farnesyltransferase from rat brain is composed of tightly associated alpha- and beta-subunits of 377 and 437 amino acids that migrate on SDS-polyacrylamide gels with apparent molecular masses of 49 and 46 kDa, respectively. The enzyme attaches farnesyl groups to cysteines in p21ras and other proteins that contain cysteine residues at the fourth position from the COOH terminus. Production of stable enzyme in animal cells requires the simultaneous synthesis of both subunits, and all activity is lost when the subunits are dissociated chemically. The beta-subunit functions in the Zn(2+)-dependent binding of the protein substrate. The role of the alpha-subunit is unknown. In the current studies we used in vitro mutagenesis and transfection of cloned cDNAs to define the parts of the alpha-subunit that are necessary to stabilize the beta-subunit and to support farnesyl transfer. Deletion of 51 amino acids at the NH2 terminus of the alpha-subunit allowed normal stabilization of the beta-subunit and production of normal enzyme activity, but deletion of 106 amino acids abolished both of these properties. A proline-rich region at residues 12-34 of the alpha-subunit is not required for activity, but its presence explains the anomalously slow migration of the polypeptide on SDS-polyacrylamide gels. Deletion of only 5 amino acids at the COOH terminus of the alpha-subunit reduced activity appreciably. Substitution of asparagine for a conserved lysine at position 164 produced an alpha-subunit that stabilized the beta-subunit normally and permitted normal binding of the two substrates, farnesyl pyrophosphate and p21H-ras. Nevertheless, the rate of transfer of the bound farnesyl group to p21H-ras was markedly reduced. The latter finding suggests that the alpha-subunit plays a direct role in the catalytic reaction in addition to its role in the stabilization of the beta-subunit.

Alkyl and Aryl Transferases↗
PubMed1993

Oocyte maturation in starfish is mediated by the beta gamma-subunit complex of a G-protein.

The stimulation of meiotic maturation of starfish oocytes by the hormone 1-methyladenine is mimicked by injection of beta gamma subunits of G-proteins from either retina or brain. Conversely, the hormone response is inhibited by injection of the GDP-bound forms of alpha i1 or alpha t subunits, or by injection of phosducin; all of these proteins should bind free beta gamma. alpha-subunit forms with reduced affinity for beta gamma (alpha i1 or alpha t bound to hydrolysis-resistant GTP analogs, or alpha i1-GMPPCP treated with trypsin to remove the amino terminus of the protein) are less effective inhibitors of 1-methyladenine action. These results indicate that the beta gamma subunit of a G-protein mediates 1-methyladenine stimulation of oocyte maturation.

Adenine↗
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

Studies on the interaction of alpha subunits of GTP-binding proteins with beta gamma dimers.

The interaction of several preparations of purified beta gamma dimers with two types of guanosine-nucleotide-binding-regulatory-(G)-protein alpha subunits, a recombinant bv alpha i3, made in Sf9 Spodoptera frugiperda cells by the baculovirus (bv) expression system, and alpha s, either purified from human erythrocyte Gs-type GTP-binding protein, and activated by NaF/AlCl3, or unpurified as found in a natural membrane, were studied. The beta gamma dimers used were from bovine rod outer segments (ROS), bovine brain, human erythrocytes (hRBC) and human placenta and contained distinct ratios of beta subunits that, upon electrophoresis, migrated as two bands with approximate M(r) of 35,000 and 36,000, as well as distinct complements of at least two gamma subunits each. When tested for their ability to recombine at submaximal concentrations with bv alpha i3, ROS, brain, hRBC and placental beta gamma dimers exhibited apparent affinities that were the same within a factor of two. When bovine brain, placental and ROS beta gamma dimers were tested for their ability to promote deactivation of Gs, brain and placental beta gamma dimers were equipotent and at least 10-fold more potent than that of ROS beta gamma dimers; likewise, brain beta gamma and placental dimers were equipotent in inhibiting GTP-activated and GTP-plus-isoproterenol-activated adenylyl cyclase, while ROS beta gamma dimers were less potent when assayed at the same concentration. The possibility that different alpha subunits may distinguish subsets of beta gamma dimers from a single cell was investigated by analyzing the beta gamma composition of three G proteins, Gs, Gi2 and Gi3, purified to near homogeneity from a single cell type, the human erythrocyte. No evidence for an alpha-subunit-specific difference in beta gamma composition was found. These findings suggests that, in most cells, alpha subunits interact indistinctly with a common pool of beta gamma dimers. However, since at least one beta gamma preparation (ROS) showed unique behavior, it is clear that there may be mechanisms by which some combinations of beta gamma dimers may exhibit selectivity for the alpha subunits they interact with.

Adenosine Diphosphate Ribose↗
PubMed1992

Characterization of guanosine diphospho-D-mannose dehydrogenase from Pseudomonas aeruginosa. Structural analysis by limited proteolysis.

Alginate is believed to be a major virulence factor in the pathogenicity of Pseudomonas aeruginosa in the lungs of patients suffering from cystic fibrosis. Guanosine diphospho-D-mannose dehydrogenase (GDPmannose dehydrogenase, EC 1.1.1.132) is a key enzyme in the alginate biosynthetic pathway which catalyzes the oxidation of guanosine diphospho-D-mannose (GDP-D-mannose) to GDP-D-mannuronic acid. In this paper, we report the structural analysis of GMD by limited proteolysis using three different proteases, trypsin, submaxillary Arg-C protease, and chymotrypsin. Treatment of GMD with these proteases indicated that the amino-terminal part of this enzyme may fold into a structural domain with an apparent molecular mass of 25-26 kDa. Multiple proteolytic cleavage sites existed at the carboxyl-terminal end of this domain, indicating that this segment may represent an exposed region of the protein. Initial proteolysis also generated a carboxyl-terminal fragment with an apparent molecular mass of 16-17 kDa which was further digested into smaller fragments by trypsin and chymotrypsin. The proteolytic cleavage sites were localized by partial amino-terminal sequencing of the peptide fragments. Arg-295 was identified as the initial cleavage site for trypsin and Tyr-278 for chymotrypsin. Catalytic activity of GMD was totally abolished by the initial cleavage. However, binding of the substrate, GDP-D-mannose, increased stability toward proteolysis and inhibited the loss of enzyme activity. GMP and GDP (guanosine 5'-mono- and diphosphates) also blocked the initial cleavage, but NAD and mannose showed no effect. These results suggest that binding of the guanosine moiety at the catalytic site of GMD may induce a conformational change that reduces the accessibility of the cleavage sites to proteases. Binding of [14C]GDP-D-mannose to the amino-terminal domain was not affected by the removal of the carboxyl-terminal 16-kDa fragment. Furthermore, photoaffinity labeling of GMD with [32P]arylazido-beta-alanine-NAD followed by proteolysis demonstrated that the radioactive NAD was covalently linked to the amino-terminal domain. These observations imply that the amino-terminal domain (25-26 kDa) contains both the substrate and cofactor binding sites. However, the carboxyl-terminal fragment (16-17 kDa) may possess amino acid residues essential for catalysis. Thus, proteolysis had little effect on substrate binding, but totally eliminated catalysis. These biochemical data are in complete agreement with amino acid sequence analysis for the existence of substrate and cofactor sites of GMD. A linear peptide map of GMD was constructed for future structure/functional studies.

Affinity Labels↗
PubMed1992

Effect of adenine metabolites on survival of Drosophila melanogaster of low xanthine dehydrogenase activity.

1. Low xanthine dehydrogenase (LXD) mutant Drosophila melanogaster were fed 0.2% adenine for 7 generations, no adenine for the next 2 generations (relaxed) and 0.2% adenine again for the next 3 generations (rechallenged) to obtain adenine-resistant lines of Drosophila (LXD-adenine). Flies grown without adenine served as LXD-controls. 2. Purines ranked as follows; adenine > adenosine > AMP > inosine > IMP in decreasing order of toxicity to LXD-adenine flies. 3. Addition of ribose to 9N position, or phosphate or carboxy to 6C position of the purine ring alleviated the toxicity. 4. More LXD-adenine offspring survived than did LXD-control offspring rechallenged with adenine.

Adenine↗
PubMed1992

Translocation of oxysterol binding protein to Golgi apparatus triggered by ligand binding.

A cDNA encoding a cytoplasmic oxysterol binding protein was expressed at high levels by transfection in animal cells. This protein binds oxysterols such as 25-hydroxycholesterol that regulate sterol metabolism by transcriptional and posttranscriptional effects. In the transfected cells, some of the oxysterol binding protein (OSBP) was distributed diffusely in the cytoplasm, and some was bound to small vesicles near the nucleus, as revealed by indirect immunofluorescence. Upon addition of 25-hydroxycholesterol, most of the OSBP became concentrated in large perinuclear structures that stained with lentil lectin, a protein that stains the Golgi apparatus. The structures that contained OSBP were disrupted by brefeldin A, confirming their identification as Golgi. A mutant OSBP lacking the COOH-terminal oxysterol binding domain localized to the Golgi spontaneously, suggesting that this domain normally occludes the domain that binds to the Golgi and that sterols relieve this occlusion. The previously noted potential leucine zipper sequence in OSBP was not required for Golgi localization, nor was it essential for homodimer formation. We conclude that OSBP is triggered to bind extrinsically to Golgi membranes when it binds oxysterols and speculate that this translocation may play a role in the transport, metabolism, or regulatory actions of oxysterols.

Animals↗
PubMed1992

The effect of transforming growth factor-alpha on airway angiogenesis.

A problem in lung transplantation is tracheal or bronchial dehiscence from ischemia. To determine if an angiogenic factor applied to the airway would improve capillary regrowth, a three-ring segment of trachea was completely severed and sutured in rats. In one group of animals the ischemic segment was wrapped with Gelfoam soaked in an angiogenic factor, transforming growth factor-alpha. In a second group the ischemic area was wrapped with Gelfoam soaked with only the vehicle. In a third group the devascularized area received no additional treatment. One animal from each group was killed daily for 7 days after operation. The tracheal vasculature was cast and viewed by light and scanning electron microscopy. None of the four animals that died early were in the transforming growth factor-alpha group. All animals lost weight between the day of operation and death, but this was least in the transforming growth factor-alpha group (p = 0.05). The light microscopy showed ischemic changes and the development of granulation tissue. The scanning electron microscopy of the vascular casts showed extensive loss of the vessels in the cut area. On day 1 the vessels of all animals dilated and their walls became rough. By day 3 a few corkscrew vessels penetrated the ischemic zone. By day 4 the animal that received transforming growth factor-alpha had more capillaries than the others. By day 6 revascularization in the transforming growth factor-alpha animal was abundant. Besides budding, new capillaries appeared to develop by lateral growth. After the fifth day vessels about 30 to 50 microns in diameter bulged focally. On the bulges, ridges the size, shape, and pattern of capillaries formed. Capillary formation in this manner has not been reported previously. Revascularization emerged sooner and more extensively with transforming growth factor-alpha. No adverse effect of transforming growth factor-alpha was found.

Animals↗