Biomedical subjects

C Yang

Publications and source records attributed to C Yang.

At least 37 records · Page 2Linked to original sources

PubMed1999

Detection of phylogenetically diverse human immunodeficiency virus type 1 groups M and O from plasma by using highly sensitive and specific generic primers.

The high degree of genetic diversity within human immunodeficiency virus type 1 (HIV-1), which includes two major groups, M (major) and O (outlier), and various env subtypes within group M (subtypes A to J), has made designing assays that will detect all known HIV-1 strains difficult. We have developed a generic primer set based on the conserved immunodominant region of transmembrane protein gp41 that can reliably amplify as few as 10 copies/PCR of viral DNA from near-full-length clones representing group M subtypes A to H (subtypes I and J were not available). The assay is highly sensitive in detecting plasma viral RNA from HIV-1 strains of diverse geographic origins representing different subtypes of HIV-1 group M as well as HIV-1 group O. Of the 253 group M plasma specimens (subtypes A, 68 specimens; B, 71; C, 19; D, 27; E, 23; F, 33; and G, 12), 250 (98.8%) were amplified by using the gp41 M/O primer set. More importantly, all 32 (100%) group O plasma samples were also amplified with these primers. In vitro spiking experiments further revealed that the assay could reliably detect as few as 25 copies/ml of viral RNA and gave positive signals in HIV-1-seropositive specimens with plasma copy numbers below the limits of detection by all commercially available viral load assays. In addition, analysis of five seroconversion panels indicated that the assay is highly sensitive for early detection of plasma viremia during the "window period." Thus, the highly sensitive assay will be useful for early detection of HIV-1 in clinical specimens from all known HIV-1 infections, regardless of their genotypes and geographic origins.

Acquired Immunodeficiency Syndrome↗

PubMed1999

Spatial coherence of forward-scattered light in a turbid medium.

We study spatially coherent forward-scattered light propagating in a turbid medium of moderate optical depth (0-9 mean free paths). Coherent detection was achieved by using a tilted heterodyne geometry, which desensitizes coherent detection of the attenuated incident light. We show that the degree of spatial coherence is significantly higher for light scattered only once in comparison with that for multiply scattered light and that it approaches a small constant value for large numbers of scattering events.

Extraterrestrial Environment↗

PubMed1999

Testing the efficacy of a recombinant merozoite surface protein (MSP-1(19) of Plasmodium vivax in Saimiri boliviensis monkeys.

Saimiri boliviensis monkeys were immunized with the yeast-expressed recombinant protein yP2P30Pv200(19). The antigen consisted of the C-terminus (amino acid Asn1622-Ser1729) of the merozoite surface protein 1 of the Plasmodium vivax Salvador I strain. Two universal T helper cell epitopes (P2 and P30) of tetanus toxin and six histidine residues for purification purposes were attached to the N- and C-termini, respectively. Four groups of five monkeys were given three immunizations at four-week intervals with either 250 microg of yP2P30Pv200(19) formulated with nonionic block copolymer P1005, 250 microg of antigen adsorbed to alum, 250 microg of antigen in phosphate-buffered saline (PBS), or PBS alone. Five weeks after the last immunization, each animal was inoculated with 100,000 parasitized erythrocytes of the Salvador I strain of P. vivax. Animals were splenectomized one week after challenge to increase parasite densities; after seven weeks of infection, animals were treated. Eighteen weeks later, the animals were rechallenged with the homologous parasite. Following the first challenge, three monkeys immunized with the antigen with P1005 were protected; no animals were protected from rechallenge. One monkey immunized with yP2P30Pv200(19) with alum was protected; no protection was seen after rechallenge. Two monkeys immunized with antigen alone were protected; none were protected from rechallenge. One control animal had a low parasite count following primary infection; none were protected against rechallenge. Adverse reactions were only observed with animals receiving P1005. It is proposed that splenectomy of the monkeys prevented adequate assessment of the efficacy of this antigen. Identification of a monkey host that supports high density parasitemia without splenectomy appears needed before further testing of blood-stage vaccines against P. vivax.

Adjuvants, Immunologic↗

PubMed1999

Role of eicosanoids in the pathogenesis of murine cerebral malaria.

Because microvascular damage is a common feature of cerebral malaria, we have examined the role eicosanoid metabolites (prostaglandins and leukotrienes) in experimental cerebral malaria. Eighty ICR mice were infected with Plasmodium berghei ANKA, with 40 uninfected mice as controls. Half of the infected mice were treated on days 4 and 5 with aspirin, a prostaglandin synthesis inhibitor. Infected mice started to die of cerebral malaria on day 6, and by day 17, all infected mice died. In contrast, all infected mice treated with aspirin died by day 12. Infected mice had increased phospholipase A2 mRNA expression in the spleen and cyclooxygenase 1 (COX1) and COX2 expression in the brain. At the peak of cerebral malaria, infected mice had higher serum leukotriene B4 levels than control mice, and aspirin-treated infected mice had higher serum leukotriene B4 levels than untreated infected mice. These results suggest that prostaglandins are protective whereas leukotrienes are detrimental in cerebral malaria.

Animals↗

PubMed1999

Cytokine production in rhesus monkeys infected with Plasmodium coatneyi.

Plasmodium coatneyi infection in rhesus monkeys has been used as a model for studying human malaria. Cytokine production in this model, however, has so far not been examined. In this study, four rhesus monkeys were infected with P. coatneyi, with another four animals serving as uninfected controls. Blood samples were taken for the determination of daily parasitemia, and cytokine and prostaglandin E2 (PGE2) levels at days 0, 3, 5, 7, and 10. All inoculated animals became infected, with synchronized appearance of ring-stage parasites. Infected monkeys had increased plasma levels of proinflammatory cytokines (interleukin-1beta, interferon-gamma, and tumor necrosis factor-alpha) during the late stage of the infection. They also had increased production of ciliary neurotrophic factor. In conjunction with the production of proinflammatory cytokines, infected monkeys also had gradual increases in the production of PGE2. A continued definition of the P. coatneyi/rhesus monkey animal model should be useful for the elucidation of the immunopathogenesis of human malaria.

Animals↗

PubMed1999

The physiological response of thrombopoietin (c-Mpl ligand) to thrombocytopenia in the rat.

It has been suggested that circulating levels of thrombopoietin (TPO) are determined primarily by platelet and megakaryocyte clearance of TPO and not by changes in hepatic TPO production. The experimental evidence accumulated so far to support this hypothesis is incomplete. We have therefore developed a new model of non-immune thrombocytopenia in the rat and used it to assess the relationship of TPO (c-mpl ligand) to the platelet mass. 14 d following the administration of busulphan, the platelet count reached a nadir of <2% of its initial value and remained at this level for up to 6 d. Circulating TPO was measured by two different bioassays which were sensitive enough to measure normal levels of TPO and levels rose from 106 +/- 29 pg/ml in animals with a normal platelet count to 2015 +/- 544 pg/ml in those with thrombocytopenia. These elevated levels of TPO were solely a response to the low platelet count since transfusion of a normal mass of platelets into the thrombocytopenic animals returned the TPO levels exactly to normal. The increase in TPO levels in thrombocytopenic animals was not due to increased TPO production since the thrombocytopenic animals did not show any increase in TPO mRNA in total or polysome-associated hepatic RNA. Rather, rat platelets were able to bind and stoichiometrically remove TPO from thrombocytopenic plasma via high-affinity receptors (Kd = 38 +/- 10 pm; 233 +/- 32 receptors/platelet). These results serve as a proof that the circulating level of TPO is determined not by alterations in TPO transcription or translation but by the ability of the platelet mass to bind and remove TPO from the circulation.

Animals↗

PubMed1999

Modifications to standard guidelines and changes in blood pressure readings: use of an automatic blood pressure device.

The purpose of the study was to determine what effect modifications to American Heart Association (AHA) guidelines produce when taking blood pressures with an automatic device. Eight blood pressure measurements were taken at least 2 minutes apart; two with standard AHA guidelines and six with modifications. Findings of this study suggest that elevation of the arm can have a significant effect on both the systolic and diastolic blood pressure. Two modifications, talking and placing the arm beside the trunk, raised the diastolic pressure. However, some modifications did not have a statistically significant effect. Techniques used to take blood pressures with an automatic device can have a significant effect on the readings.

Adult↗

PubMed1999

The expression of insulin-like growth factor II, hepatitis B virus X antigen and p21 in experimental hepatocarcinogenesis in tree shrews.

The purpose of this paper was to study the mechanism of synergistic effect in hepatocarcinogenesis induced by hepatitis B virus (HBV) infection and aflatoxin B1 (AFB1) intake. Immunohistochemical staining was used in formalin-fixed, paraffin-embedded sections of cancer and liver tissues. The incidence of hepatocellular carcinomas (HCCs) was 52.9% in experimental tree shrews that received both HBV and AFB1. It was significantly higher than that of animals exposed to HBV (11.1%, Group B), or (AFB1) (15.8%, group C) alone. HCC was not found in the control animals (group D). The expressions of insulin-like growth factor II (IGF-II) were 82.4%, 22.2%, 26.3% and 0 in groups A, B, C and D, respectively. The significant differences of IGF-II were observed between groups A and B, C and D (P < 0.05). The expressions of p21 were 29.4%, 11.1%, 15.8% and 0 in group A, B, C and D, respectively. The positive rate of hepatitis B x antigen (HbxAg) was significantly higher in the group A than that in the group B (52.9% vs. 11.1%, P < 0.05). The parallel relations between the incidence of HCC and the overexpressions of these genes protein have been found in each group. On the other hand, the expressions of these genes in tumour-bearing tree shrews were significantly higher than that in nontumour-bearing animals. These findings suggest a synergistic effects of HBV and AFB1 in activation of these genes in tree shrews. Overexpressions of these genes may take an important role in the course of hepatocarcinogenesis in tree shrews.

Aflatoxin B1↗

PubMed1999

Synergistic effect of hepatitis B virus and aflatoxin B1 in hepatocarcinogenesis in tree shrews.

An animal experiment with tree shrews was performed to detect the synergistic effects of hepatitis B virus (HBV) infection and dietary aflatoxin B1 (AFB1) in hepatocarcinogenesis. Adult healthy tree shrews (Tupaia belangeri chinensis) were divided into four groups: Group A (HBV + AFB1)--animals were infected with human HBV serum at first, then fed AFB1 diluted with milk, 150 ug/kg.bw/day, 6 days/week for 105 weeks. Group B (HBV)--animals were infected with human HBV as Group A, but no AFB1 treatment. Group C (AFB1)--animals were treated with AFB1 as Group A but no HBV infection. Group D--animals were treated neither with human HBV nor AFB1. During the experiment, blood samples and liver biopsies were taken regularly from all animals in each group. All the animals were sacrificed on the 160th week when the experiment ended. The samples of sera and liver tissues were checked for HBV markers and histological changes. Hepatocellular carcinomas (HCCs) were found only in Group A and Group C, with incidences of 67% and 30% respectively. The average time for HCC occurrence in Group A and Group C was 120.3 +/- 16.6 and 153.3 +/- 5.8 weeks respectively (P < 0.01). Even though no HCC occurred in Group B, 1 animal which died before the end of the experiment showed two large hepatocellular nodules. These results showed that there is synergistic effect between HBV and AFB1 in tree shrews' hepatocarcinogenesis, even though the hepatocarcinogenic effect played by HBV alone is rather weak.

Aflatoxin B1↗

PubMed1998

Modulation of aromatase expression in the breast tissue by ERR alpha-1 orphan receptor.

We have previously identified a silencer element (S1) that is situated between promoters I.3 and II of the human aromatase gene and that down-regulates the action of these promoters. We recently applied the yeast one-hybrid approach to screen a human breast tissue hybrid cDNA expression library for genes encoding the proteins binding to the silencer region. Most proteins identified from this approach belong to the nuclear receptor superfamily. Fifty % of the positive clones encode for ERR alpha-1, and other positive clones include EAR-2, EAR-3 (COUP-TF1), RAR gamma, and p120E4F. Because ERR alpha-1 was found to be the major protein interacting with S1, we decided to examine the regulatory action of ERR alpha-1 on promoter I.3 of the human aromatase gene. Using a reporter plasmid that includes the aromatase genomic fragment containing promoter I.3 and S1, ERR alpha-1 was found to have a positive regulatory function in breast cancer SK-BR-3 cells. Gel mobility shift assays have confirmed that ERR alpha-1 binds to S1 in a dose-dependent manner, and DNase I footprinting analysis has revealed that ERR alpha-1 binds to a region, 5'-AAGGTCAGAAAT-3', which is within S1 and between 96 and 107 bp relative to the transcriptional start site of promoter I.3. In addition, despite the fact that the nuclear receptor SF1 was shown previously to bind to the same site and to mediate a cAMP response in ovary, our yeast one-hybrid screening did not find any SF-1 clones. Gel mobility shift assays further revealed that SF-1 can bind to the silencer element with an affinity comparable with ERR alpha-1. Because our reverse transcription-PCR analysis was not able to detect SF1 mRNA in breast cancer tissue or in SK-BR-3 cells, it is thought that SF1 protein is not expressed in breast cancer tissue. Two ERR alpha-1 RNA variants with differences at the 5'-end have been reported. Our reverse transcription-PCR analysis identified the shorter variant in 28 of 32 breast tumor specimens and the longer variant in only 1 specimen. In addition, the shorter variant was detected in breast cancer SK-BR-3 cells as well as in a breast tumor fibroblast line WS3TF. The results suggest that ERR alpha-1 is one of the nuclear proteins interacting with S1 in breast cancer tissue. It is thought that the silencer element in the human aromatase gene may function differently in different tissues because of distinct expression patterns of transcription factors.

Aromatase↗

PubMed1998

Kinetics of Particle Transport to a Solid Surface from an Impinging Jet under Surface and External Force Fields.

Based on the presented model for the impinging jet system, extensive theoretical analysis was made on particle deposition. Complete transport equations with consideration of gravity, van der Waals, and electrical double layer (EDL) interactions, as well as hydrodynamic interactions, were numerically solved. The influences of gravity, van der Waals, and electrical double layer interactions on the particle deposition rates (in terms of the Sherwood number) were presented. The results demonstrate that the asymmetric EDL interaction, which has been ignored in previous treatments, has an impact on the particle deposition rate. It was also found that the Sherwood number is strongly dependent on the characteristics of the particle-collector interaction energy profiles, such as the height of the energy barrier and the depth of the secondary energy minimum. Particularly, the effects of the height of the energy barrier and the depth of the secondary energy minimum on the Sherwood number for different Peclet numbers were discussed. In addition, a simple expression was derived for quantitatively estimating the contributions to the deposition rate due to particle diffusion, migration, and convection. With the aid of calculated particle concentration distributions, this expression can be used to understand the numerical predictions. Copyright 1998 Academic Press.

Journal Article↗

PubMed1998

Noradrenergic-specific transcription of the dopamine beta-hydroxylase gene requires synergy of multiple cis-acting elements including at least two Phox2a-binding sites.

Dopamine beta-hydroxylase (DBH) catalyzes the conversion of dopamine to noradrenaline and is selectively expressed in noradrenergic and adrenergic neurons and neuroendocrine cells. Recent data from this laboratory showed that a paired-like homeodomain (HD) protein, Phox2a, interacts with the HD-binding site residing within a composite promoter of the human DBH gene, designated domain IV, in a cell-specific manner and directly controls noradrenergic-specific DBH promoter activity. In this report, we demonstrate that three additional protein-binding sites (i.e., domains I, II, and III) between domain IV and the TATA box are critical for intact DBH promoter activity in noradrenergic cells and that they activate DBH transcription in a highly concerted manner. Transient transfection assays of mutant DBH reporter constructs indicated that domain I was active in every cell line tested, whereas domain III was preferentially active in DBH-positive cells. Remarkably, mutation of domain II was associated with inactivation of DBH promoter activity exclusively in DBH-positive cell lines, defining it as another noradrenergic-specific promoter element. The cell-specific profile of the promoter function of these sequence motifs was further supported by in vitro DNA-binding studies and Southwestern analysis. Furthermore, competition and antibody supershift assays show that transcription factors Sp1 and AP2 are the cognate nuclear factors interacting with domains I and III, respectively. Parallel evidence indicates that domain II is another Phox2a-binding site, demonstrating at least two binding sites for this factor in the upstream DBH promoter. Strikingly, four tandem copies of domain II increased the promoter activity of a minimal DBH promoter by 100- to 200-fold in DBH-positive cell lines without compromising cell specificity. Cotransfection of Phox2a-expression vector dramatically increased the activity of the multiple domain II promoter only in DBH-negative cell lines, confirming that domain II is responsive to Phox2a. Collectively, this study emphasizes a critical role of Phox2a as well as its functional synergism with other transcription factors (e.g., CREB, AP2, and Sp1) in transcriptional activation of the DBH gene.

Animals↗

PubMed1998

Disulfide isomerization within the C-terminus of cobrotoxin decelerates by thiol compounds and trinitrophenylation, but accelerates by modification of carboxyl groups.

A disulfide isomerization at the C-terminus of cobrotoxin occurred spontaneously by dissolving in alkali buffer. Irreversible conversion of cobrotoxin into its isomers was completely achieved within 4 days. The isomerization reaction was decelerated by thiol compounds including GSSG, GSH, cystamine, and cysteamine in a pseudo-first-order kinetic, and GSSG was the most effective one among the thiol compounds used. Moreover, the oxidized thiol compounds were always superior to reduced ones in decreasing the rate of disulfide interchange. To further assess the intrinsic elements essential for the occurrence of disulfide isomerization of cobrotoxin, the toxin molecule was subjected to modification on its Arg, Lys, Trp, Tyr, and carboxyl groups. In sharp contrast to other modified derivatives, the isomerization reaction was decelerated by trinitrophenylation on Lys-26, Lys-27, and Lys-47, whereas it was rapidly completed after modification of carboxyl groups. Neither chemical modification nor the toxin's conformation affected the irreversibility of isomerization reaction. Thus, the observed change in the rate of disulfide isomerization reflects the involvement of Lys residues and carboxyl groups in this reaction. Although thiol compounds further decelerated the conversion of trinitrophenylated cobrotoxin into its isomers, they did not exert a notable effect on the isomerization of carboxyl groups-modified derivative. These results clearly indicate that disulfide isomerization of cobrotoxin is, in part, driven by the positively charged Lys residues at positions 26, 27, and 47 of the toxin molecule, and that the thiol compounds are coordinated with the negatively charged groups of cobrotoxin to exert their inhibitory action.

Animals↗

PubMed1998

Mechanism of Cdc42-induced actin polymerization in neutrophil extracts.

Cdc42, activated with GTPgammaS, induces actin polymerization in supernatants of lysed neutrophils. This polymerization, like that induced by agonists, requires elongation at filament barbed ends. To determine if creation of free barbed ends was sufficient to induce actin polymerization, free barbed ends in the form of spectrin-actin seeds or sheared F-actin filaments were added to cell supernatants. Neither induced polymerization. Furthermore, the presence of spectrin-actin seeds did not increase the rate of Cdc42-induced polymerization, suggesting that the presence of Cdc42 did not facilitate polymerization from spectrin-actin seeds such as might have been the case if Cdc42 inhibited capping or released G-actin from a sequestered pool. Electron microscopy revealed that Cdc42-induced filaments elongated rapidly, achieving a mean length greater than 1 micron in 15 s. The mean length of filaments formed from spectrin-actin seeds was <0.4 micron. Had spectrin-actin seeds elongated at comparable rates before they were capped, they would have induced longer filaments. There was little change in mean length of Cdc42-induced filaments between 15 s and 5 min, suggesting that the increase in F-actin over this time was due to an increase in filament number. These data suggest that Cdc42 induction of actin polymerization requires both creation of free barbed ends and facilitated elongation at these ends.

Actin Cytoskeleton↗

PubMed1998

Identification of a novel type of silk protein and regulation of its expression.

The silk of lepidopteran insects has been studied extensively as proteins of two categories: the fibroins, which are produced in the posterior section of silk glands, and the sericins, which are secreted in the middle section. We now describe a third category that is named seroins to accentuate the fact that both the sericin- and the fibroin-producing cells participate in seroin secretion. Using a probe derived from the N-terminal sequences of a 23-kDa components of Galleria mellonella silk, we isolated silk gland-specific cDNA encoding 167 amino acids, of which 17 constitute the signal peptide. The following 14 residues match the N-terminal sequences of the 23- and 22.5-kDa silk proteines. The reaction of these proteins with concanavalin A and the presence of two glycosylation sites in the seroin peptide sequence indicate that seroin is secreted in two forms that both contain a mannose-rich sugar moiety. Seroin is distinguished from other silk proteins by high proline content (34 residues or 20.26% by weight), lack of cysteines, and the presence of two kinds of short amino acid repeats. The seroin gene is expressed in both the posterior and middle silk gland sections. The expression fluctuates during development in correlation with the feeding regime and the changes in hormone titers: seroin mRNA is high in the silk glands of feeding larvae, declines at ecdysis, reaches a maximum during cocoon spinning, and thereafter rapidly drops to an undetectable level. In vivo and in vitro experiments showed that the drop is caused by ecdysteroid hormones and is prevented by juvenile hormones. N-terminal sequencing of several silk proteins of Bombyx mori revealed that the 8- and 13-kDa proteins share 5 or 6 out of 10 identified amino acids with the N terminus of Galleria seroin and obviously represent seroin homologues. The result suggests that seroin-type proteins are a general component of lepidopteran silk.

Amino Acid Sequence↗

PubMed1998

Mcm1 regulates donor preference controlled by the recombination enhancer in Saccharomyces mating-type switching.

Switching of Saccharomyces mating type by replacement of sequences at the MAT locus involves a choice between two donors, HML and HMR. MATalpha cells inhibit recombination along the entire left arm of chromosome III, including HML, whereas MATa cells activate this same region. MATa-dependent activation of HML depends on a small, cis-acting DNA sequence designated the recombination enhancer (RE), located 17 kb centromere-proximal to HML. A comparison of RE sequences interchangeable between Saccharomyces cerevisiae and Saccharomyces carlsbergensis defines a minimum RE of 244 bp. RE activity is repressed in MATalpha cells by binding of the Matalpha2-Mcm1 corepressor to a site within the RE. Mutation of the two Matalpha2 binding sites removes most, but not all, of this repression, and RE chromatin structure in MATalpha cells becomes indistinguishable from that seen in MATa. Surprisingly, a 2-bp mutation in the Mcm1 binding site completely abolishes RE activity in MATa cells; moreover, RE chromatin structure in the MATa mutant becomes very similar to that seen in MATalpha cells with a normal RE, displaying highly ordered nucleosomes despite the absence of Matalpha2. Further, a mutation that alters the ability of Mcm1 to act with Matalpha2 in repressing a-specific genes also alters donor preference in either mating type. Thus, Mcm1 is critically responsible for the activation as well as the Matalpha2-Mcm1-mediated repression of RE activity.

Base Sequence↗

PubMed1998

Characterization of the P25 silk gene and associated insertion elements in Galleria mellonella.

Insect silk genes attract attention by their precise territorial and developmental regulations and extremely high expression rates. Our present investigations demonstrated that the P25 silk gene of Galleria mellonella is down-regulated by ecdysteroid hormones. The gene was identified within 5217 nucleotides (nt) of two genomic clones. In contrast to other silk genes, Galleria P25 lacks the canonical TATA box. Transcription is initiated within a region of three nucleotides that lie at the end of a capsite initiator sequence ACAGT and about 90 nt downstream from a CAAT box. A stretch of 32 nt with a core sequence CTTTT was detected in the 5' region of Galleria P25 as well as in the presumptive regulatory regions of all other silk genes that are expressed in the posterior silk gland. However, consensus sequences reported for the regulatory regions of Bombyx silk genes are not obvious in Galleria P25. The coding sequence of this gene included 654 nt, is interrupted by 4 introns, and ends in position +3369; a potential polyadenylation signal starts at +4382. The gene contains 3 copies of a short interspersed nuclear element (SINE), which are located in the upstream region (-833 to -579) and in the first (+542 to +840) and second (+2259 to +2556) introns. The repeat, which was named Gm1, occurs in some other Galleria genes and exhibits homology to Bm1 SINE of the silkworm and to a similar element of a spider. Another insertion of at least 150 nt and with loosely defined borders is present in the 3' untranslated region (UTR) of Galleria P25. It includes a box (+3453 to +3552) of 99 nt that is tentatively called Lep1 because it was disclosed also in some other Lepidoptera. Lep1 seems to represent the core region of insertion elements that occur in the genomes of lepidopteran insects in various species specific and region specific modifications.

Amino Acid Sequence↗

PubMed1998

ARF6 targets recycling vesicles to the plasma membrane: insights from an ultrastructural investigation.

We have shown previously that the ADP-ribosylation factor (ARF)-6 GTPase localizes to the plasma membrane and intracellular endosomal compartments. Expression of ARF6 mutants perturbs endosomal trafficking and the morphology of the peripheral membrane system. However, another study on the distribution of ARF6 in subcellular fractions of Chinese hamster ovary (CHO) cells suggested that ARF6 did not localize to endosomes labeled after 10 min of horseradish peroxidase (HRP) uptake, but instead was uniquely localized to the plasma membrane, and that its reported endosomal localization may have been a result of overexpression. Here we demonstrate that at the lowest detectable levels of protein expression by cryoimmunogold electron microscopy, ARF6 localized predominantly to an intracellular compartment at the pericentriolar region of the cell. The ARF6-labeled vesicles were partially accessible to HRP only on prolonged exposure to the endocytic tracer but did not localize to early endocytic structures that labeled with HRP shortly after uptake. Furthermore, we have shown that the ARF6-containing intracellular compartment partially colocalized with transferrin receptors and cellubrevin and morphologically resembled the recycling endocytic compartment previously described in CHO cells. HRP labeling in cells expressing ARF6(Q67L), a GTP-bound mutant of ARF6, was restricted to small peripheral vesicles, whereas the mutant protein was enriched on plasma membrane invaginations. On the other hand, expression of ARF6(T27N), a mutant of ARF6 defective in GDP binding, resulted in an accumulation of perinuclear ARF6-positive vesicles that partially colocalized with HRP on prolonged exposure to the tracer. Taken together, our findings suggest that ARF activation is required for the targeted delivery of ARF6-positive, recycling endosomal vesicles to the plasma membrane.

ADP-Ribosylation Factor 6↗