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PubMed2002

Yeast Ysl2p, homologous to Sec7 domain guanine nucleotide exchange factors, functions in endocytosis and maintenance of vacuole integrity and interacts with the Arf-Like small GTPase Arl1p.

We previously described the isolation of ysl2-1 due to its genetic interaction with Delta ypt51/vps21, a mutant with a deletion of the coding sequence for the yeast Rab5 homolog, which regulates endocytic traffic between early and late endosomes. Here we report that Ysl2p is a novel 186.8-kDa peripheral membrane protein homologous to members of the Sec7 family. We provide multiple genetic and biochemical evidence for an interaction between Ysl12p and the Arf-like protein Arl1p, consistent with a potential function as an Arf guanine nucleotide exchange factor (GEF). The temperature-sensitive alleles ysl2-307 and ysl2-316 are specifically defective in ligand-induced degradation of Ste2p and alpha-factor and exhibit vacuole fragmentation directly upon a shift to 37 degrees C. In living cells, green fluorescent protein (GFP)-Ysl2p colocalizes with endocytic elements that accumulate FM4-64. The GFP-Ysl2p staining is sensitive to a mutation in VPS27 resulting in the formation of an aberrant class E compartment, but it is not affected by a sec7 mutation. Consistent with the idea that Ysl2p and Arl1p have closely related functions, Delta arl1 cells are defective in endocytic transport and in vacuolar protein sorting.

ADP-Ribosylation Factors↗
PubMed2002

Obligate roles for p16(Ink4a) and p19(Arf)-p53 in the suppression of murine pancreatic neoplasia.

Epithelial tumors of the pancreas exhibit a wide spectrum of histologies with varying propensities for metastasis and tissue invasion. The histogenic relationship among these tumor types is not well established; moreover, the specific role of genetic lesions in the progression of these malignancies is largely undefined. Transgenic mice with ectopic expression of transforming growth factor alpha (TGF-alpha) in the pancreatic acinar cells develop tubular metaplasia, a potential premalignant lesion of the pancreatic ductal epithelium. To evaluate the cooperative interactions between TGF-alpha and signature mutations in pancreatic tumor genesis and progression, TGFalpha transgenic mice were crossed onto Ink4a/Arf and/or p53 mutant backgrounds. These compound mutant mice developed a novel pancreatic neoplasm, serous cystadenoma (SCA), presenting as large epithelial tumors bearing conspicuous gross and histological resemblances to their human counterpart. TGFalpha animals heterozygous for both the Ink4a/Arf and the p53 mutation showed a dramatically increased incidence of SCA, indicating synergistic interaction of these alleles. Inactivation of p16(Ink4a) by loss of heterozygosity, intragenic mutation, or promoter hypermethylation was a common feature in these SCAs, and correspondingly, none of the tumors expressed wild-type p16(Ink4a). All tumors sustained loss of p53 or Arf, generally in a mutually exclusive fashion. The tumor incidence data and molecular profiles establish a pathogenic role for the dual inactivation of p16(Ink4a) and p19(Arf)-p53 in the development of SCA in mice, demonstrating that p16(Ink4a) is a murine tumor suppressor. This genetically defined model provides insights into the molecular pathogenesis of SCA and serves as a platform for dissection of cell-specific programs of epithelial tumor suppression.

Animals↗
PubMed2001

Temperature measurement for energy-efficient ablation by thermal radiation with a microsecond time constant from the corneal surface during ArF excimer laser ablation.

Measurement of the temperature of the corneal surface during photorefractive keratectomy (PRK) is thought to be useful for monitoring the corneal ablation process, since the photothermal process has been proposed as the major mechanism of ArF excimer laser ablation. For temperature measurement, we measured thermal radiation from the corneal surface during ArF excimer laser ablation using a mercury-cadmium-telluride detector with a 1-micros time constant. To investigate the effects of temperature on ablation depth, the ablation depth of the cornea was measured by microscopy. When corneal ablation was initiated at the fluence of 65 mJ/cm2, the corneal surface temperature rose to 60-70 degrees C. The energy required for a unit-depth ablation (degrees C/microm) was lowest at 120 micro C. Monitoring of transient temperature during PRK provides important information on energy-efficient ablation, which may enable rapid and safe corneal incisions.

Animals↗
PubMed2002

Effect of amylase-rich flour (ARF) treatment on the viscosity of fermented complementary foods.

Grains of cowpea and maize and slices of fresh cassava, cocoyam, plantain, and yam were steep-fermented in water, while flours from the same plant materials were fermented by backslopping for 24 to 30 hours. The pH and apparent viscosity of the gruels from the resulting flours were determined. Loss in weight due to fermentation was higher in fresh tubers than in dry grains. The pH of the flours decreased during fermentation. Measurements showed that the apparent viscosity only of gruels from flours produced by backslopping decreased after 24 hours. The apparent viscosity of gruels from steep-fermented flours was higher than that of the unfermented flours and those produced by backslopping. The apparent viscosity reduction of gruels from steep-fermented flours using amylase-rich flour (ARF) from five-day white sorghum malt was better with the prepared gruels than when applied to the flour-in-tap-water suspension before it was used to prepare gruels. Viscosity reduction using ARF was also better with the fermented gruels than with gruels from unfermented flours. The implications of these results for the formulation of complementary flour blends for infant feeding are discussed.

Amylases↗
PubMed1996

The mammalian ARF-like protein 1 (Arl1) is associated with the Golgi complex.

A rat cDNA clone was isolated which encodes a protein displaying characteristics of a ras-like small GTPase. The deduced amino acid sequence shows the highest amino acid identity (79%) with the Drosophila ARF-like protein 1 (dArl1) among all the known members of the ras-like small GTPase superfamily. The encoded protein was tentatively named rat Arl1 (rArl1). Northern blotting analysis revealed that the rArl1 gene is ubiquitously expressed in rat tissues. Recombinant rArl1 fused to glutathione-S-transferase (GST) to create GST-rArl1 binds GTP-gamma-S in a dose-dependent manner. Polyclonal antibodies raised against two unique rArl1 peptides recognized a 22 kDa protein in total NRK cell lysate. Immunofluorescence microscopy of NRK cells revealed discrete perinuclear labelling that could be competed out by GST-rArl1 but not GST. Examination of 8 additional cell lines revealed a similar labelling, suggesting that the antigen recognised by the antibodies is conserved and widely-expressed. Co-localization experiments in NRK cells with antibodies to mannosidase II and a newly identified cis-Golgi protein, p28, showed that rArl1 is localized to the Golgi complex. When cells were treated with nocodazole, the Golgi complex marked by mannosidase II and p28 was fragmented into punctate structures scattered throughout the cell, in which rArl1 was colocalized. Treatment with brefeldin A (BFA) resulted in the redistribution of rArl1 and mannosidase II into the cytoplasm and endoplasmic reticulum, respectively. The kinetics of the redistribution of rArl1 in response to BFA differ from those of ARF and beta-COP, two components of non-clathrin coated vesicles.

ADP-Ribosylation Factors↗
PubMed2001

The ARF exchange factors Gea1p and Gea2p regulate Golgi structure and function in yeast.

The Sec7 domain guanine nucleotide exchange factors (GEFs) for the GTPase ARF are highly conserved regulators of membrane dynamics. Their precise molecular roles in different trafficking steps within the cell have not been elucidated. We present a functional analysis of two members of this family, Gea1p and Gea2p, in the yeast Saccharomyces cerevisiae. Gea1p and Gea2p can functionally replace each other, but at least one is necessary for viability. Temperature sensitive gea mutants were generated and found to have defects in ER-Golgi and intra-Golgi transport. Similar to mutants in COPI subunits in yeast, gea mutants had a cargo-selective secretion defect, in that some proteins continued to be secreted whereas others were blocked in the ER or early Golgi. Like yeast arf mutants, the rate of transport of those proteins that continued to be secreted was slowed. In addition, the structure of Golgi elements was severely perturbed in gea mutants. We conclude that Gea1p and Gea2p play an important role in the structure and functioning of the Golgi apparatus in yeast.

ADP-Ribosylation Factors↗
PubMed2001

Regulation of Golgi structure and function by ARF-like protein 1 (Arl1).

Arl1 is a member of the ARF-like protein (Arl) subfamily of small GTPases. Nothing is known about the function of Arl1 except for the fact that it is essential for normal development in Drosophila and that it is associated with the Golgi apparatus. In this study, we first demonstrate that Arl1 is enriched at the trans side of the Golgi, marked by AP-1. Association of Arl1 with the Golgi is saturable in intact cells and depends on N-terminal myristoylation. Over-expression of Arl1(T31N), which is expected to be restricted to the GDP-bound form and thus function as a dominant-negative mutant, causes the disappearance of the Golgi apparatus (marked by Golgi SNARE GS28), suggesting that Arl1 is necessary for maintaining normal Golgi structure. Overexpression of Arl1(Q71L), a mutant restricted primarily to the activated GTP-bound form, causes an expansion of the Golgi apparatus with massive and stable Golgi association of COPI and AP-1 coats. Interestingly, Golgi ARFs also become stably associated with the expanded Golgi. Transport of the envelope protein of vesicular stomatitis virus (VSV-G) along the secretory pathway is arrested at the expanded Golgi upon expression of Arl1(Q71L). The structure of stacked cisternae of the Golgi is disrupted in cells expressing Arl1(Q71L), resulting in the transformation of the Golgi into an extensive vesicule-tubule network. In addition, the GTP form of Arl1 interacts with arfaptin-2/POR1 but not GGA1, both of which interact with GTP-restricted ARF1, suggesting that Arl1 and ARF1 share some common effectors in regulating cellular events. On the basis of these observations, we propose that one of the mechanisms for the cell to regulate the structure and function of the Golgi apparatus is through the action of Arl1.

ADP-Ribosylation Factors↗
PubMed2006

The functional relationship between the Cdc50p-Drs2p putative aminophospholipid translocase and the Arf GAP Gcs1p in vesicle formation in the retrieval pathway from yeast early endosomes to the TGN.

Drs2p, the catalytic subunit of the Cdc50p-Drs2p putative aminophospholipid translocase, has been implicated in conjunction with the Arf1 signaling pathway in the formation of clathrin-coated vesicles (CCVs) from the TGN. Herein, we searched for Arf regulator genes whose mutations were synthetically lethal with cdc50Delta, and identified the Arf GAP gene GCS1. Most of the examined transport pathways in the Cdc50p-depleted gcs1Delta mutant were nearly normal, including endocytic transport to vacuoles, carboxypeptidase Y sorting, and the processing and secretion of invertase. In contrast, this mutant exhibited severe defects in the early endosome-to-TGN transport pathway; proteins that are transported via this pathway, such as the v-SNARE Snc1p, the t-SNARE Tlg1p, and the chitin synthase III subunit Chs3p, accumulated in TGN-independent aberrant membrane structures. We extended our analyses to clathrin adaptors, and found that Gga1p/Gga2p and AP-1 were also involved in this pathway. The Cdc50p-depleted gga1Delta gga2Delta mutant and the gcs1Delta apl2Delta (the beta1 subunit of AP-1) mutant exhibited growth defects and intracellular Snc1p-containing membranes accumulated in these cells. These results suggest that Cdc50p-Drs2p plays an important role in the Arf1p-mediated formation of CCVs for the retrieval pathway from early endosomes to the TGN.

ADP-Ribosylation Factor 1↗
PubMed2005

Expression of ADP-ribosylation factor (ARF)-like protein 6 during mouse embryonic development.

ADP-ribosylation factor (ARF)-like protein 6 (ARL6) is a member of the ARF-like protein (ARL) subfamily of small GTPases (Moss, 1995; Chavrier, 1999). ARLs are highly conserved through evolution and most of them possess the consensus sequence required for GTP binding and hydrolysis (Pasquallato, 2002). Among ARLs, ARL6 which was initially isolated from a J2E erythroleukemic cell line is divergent in its consensus sequences and its expression has been shown to be limited to the brain and kidney in adult mouse (Ingley, 1999). Recently, it was reported that mutations of the ARL6 gene cause type 3 Bardet-Biedl syndrome in humans and that ARL6 is involved in ciliary transport in C. elegans (Chiang, 2004; Fan, 2004). Here, we investigated the expression pattern of ARL6 during early mouse development by whole-mount in situ hybridization and found that interestingly, ARL6 mRNA was localized around the node at 7.0-7.5 days post coitum (dpc) embryos, while weak expression was also found in the ectoderm. At the later stage (8.5 dpc) ARL6 was expressed in the neural plate and probably in the somites. Based on these results, a possible role of ARL6 in early development is discussed in relation to the findings in human and C. elegans (Chiang, 2004; Fan, 2004).

ADP-Ribosylation Factors↗
PubMed2004

N-Terminal polyubiquitination of the ARF tumor suppressor, a natural lysine-less protein.

Ubiquitin-dependent proteolysis by proteasomes generally depends upon the conjugation of polyubiquitin chains to lysine epsilon-NH(2) groups within the targeted proteins. However, engineered lysine-less mutants of certain viral and cellular proteins can undergo polyubiquitination at their N-termini. Is N-terminal polyubiquitination a physiologic process, and how many cellular proteins can be targeted for proteasomal degradation through this mechanism? Recent work indicates that the turnover of the endogenous lysine-less human ARF tumor suppressor protein and its mouse Arf counterpart (containing a single, non-conserved lysine residue) is regulated in this manner.

Cyclin-Dependent Kinase Inhibitor p16↗
PubMed2005

Modulation of the ARF-p53 pathway by the small DNA tumor viruses.

The small DNA tumor viruses encode proteins that subvert many of the pivotal growth regulatory pathways within the cell to facilitate their own replication. The cell responds to viral infection/proteins by activating the p53 tumor suppressor pathway. Activation of p53 could impair a productive viral infection at many levels, including the inhibition of viral DNA replication and/or the premature death of infected cells. Therefore, DNA viruses encode proteins that inactivate the p53 tumor suppressor pathway. Understanding how DNA viral proteins activate/inactivate the p53 pathway has provided invaluable insights into tumorigenesis. Recent studies with polyoma virus have identified a viral protein (PyST) that inhibits ARF-mediated activation of p53, and revealed a novel role for PP2A in the regulation of the ARF-p53 tumor suppressor pathway.

ADP-Ribosylation Factor 1↗
PubMed1999

A role for E2F1 in the induction of ARF, p53, and apoptosis during thymic negative selection.

E2F transcriptional activity controls the expression of many of the genes required for G1 to S phase progression. E2F1, one member of the E2F family, plays an important role in the induction of apoptosis. We have examined the role of the E2F1 transcription factor in apoptosis during T-cell maturation in the thymus. We show that E2F1 is required for the apoptosis of autoimmune immature T cells during thymic negative selection in vivo. This T-cell receptor-mediated apoptosis coincides with the E2F1-dependent increase of p19-ARF mRNA and p53 protein levels. In contrast, E2F1 is not required for the induction of apoptosis by glucocorticoids or DNA damage. These results demonstrate a specific role for E2F1, which triggers a pathway leading to ARF and p53 induction, in a physiological apoptosis pathway that is uncoupled from a normal proliferative event.

Animals↗
PubMed2000

p53 stabilization and functional impairment in the absence of genetic mutation or the alteration of the p14(ARF)-MDM2 loop in ex vivo and cultured adult T-cell leukemia/lymphoma cells.

Human T-cell lymphotropic virus type I (HTLV-I) transforms T cells in vitro, and the viral transactivator Tax functionally impairs the tumor suppressor p53 protein, which is also stabilized in HTLV-I-infected T cells. Thus, the functional impairment of p53 is essential to maintain the viral-induced proliferation of CD4+ mature T cells. However, in the CD4+ leukemic cells of patients with adult T-cell leukemia/lymphoma (ATLL), the viral transactivator does not appear to be expressed, and p53 mutations have been found only in a fraction of patients. We sought to investigate whether p53 function is impaired, in ex vivo samples from patients with ATLL, in the absence of genetic mutations. Here we demonstrate that the p53 protein is stabilized also in ex vivo ATLL samples (10 of 10 studied) and that at least in 2 patients p53 stabilization was not associated with genetic mutation. Furthermore, the assessment of p53 function after ionizing radiation of ATLL cells indicated an abnormal induction of the p53-responsive genes GADD45 and p21(WAF1) in 7 of 7 patients. In 2 of 2 patients, p53 regulation of cell-cycle progression appeared to be impaired as well. Because p53 is part of a regulatory loop that also involves MDM2 and p14(ARF), the status of the latter proteins was also assessed in cultured or fresh ATLL cells. The p97 MDM2 protein was not detected by Western blot analysis in established HTLV-I-infected T-cell lines or ex vivo ATLL cell lysates. However, the MDM2 protein could be easily detected after treatment of cells with the specific proteasome inhibitor lactacystin, suggesting a normal regulation of the p53-MDM2 regulating loop. Similarly, p14(ARF) did not appear to be aberrantly expressed in ex vivo ATLL cells nor in any of the established HTLV-I-infected T-cell lines studied. Thus, p53 stabilization in HTLV-I infection occurs in the absence of genetic mutation and alteration of the physiologic degradation pathway of p53. (Blood. 2000;95:3939-3944)

Adult↗
PubMed2001

E2F-1 up-regulates c-Myc and p14(ARF) and induces apoptosis in colon cancer cells.

Although overexpression of E2F-1 can induce apoptosis in a variety of tumor cell lines, the mechanisms by which E2F-1 induces apoptosis remain ambiguous. In this study, we examine the ability of E2F-1 to induce apoptosis in colon cancer and the molecular mechanisms underlying E2F-1-mediated apoptosis. HT-29 and SW-620 colon adenocarcinoma cells (both mutant p53) were treated by mock infection or adenoviral vectors Ad5CMV (empty vector), Ad5CMVLacZ (beta-galactosidase), and Ad5CMVE2F-1 (E2F-1) at multiplicity of infection of 100. Western blot analysis confirmed marked overexpression of E2F-1 in both cell lines. By 5 days after infection, E2F-1 overexpression resulted in >25-fold reduction in cell growth and >90% loss of cell viability in both cell lines. Cell cycle analysis of Ad-E2F-1-infected cells revealed an increase in G(2)/M and sub-G(1) populations. By in situ terminal deoxynucleotidyl transferase (Tdt)-mediated nick end labeling analysis, evidence of apoptosis was observed including internucleosomal DNA fragmentation and the formation of apoptotic bodies. In addition, caspase-3 and poly(ADP-ribose) polymerase apoptotic fragments were detected by 48 h after treatment with Ad-E2F-1. Of mechanistic importance, overexpression of E2F-1 caused a G(2)/M arrest followed by increased levels of c-Myc and p14(ARF) proteins. Additionally, expression of the antiapoptotic Bcl-2 family member Mcl-1 was down-regulated in E2F-1-overexpressing cells. In conclusion, E2F-1 overexpression initiates apoptosis and suppresses growth in HT-29 and SW620 colon adenocarcinoma cells. Overexpression of E2F-1 triggers apoptosis and is associated with up-regulation of c-Myc and p14(ARF) proteins and down-regulation of Mcl-1. Therefore, E2F-1 is a potentially active gene therapy agent for the treatment of colon cancer.

Adenoviridae↗
PubMed2001

[The INK4a-ARF locus: role in the genetic predisposition to familial melanoma and in skin carcinogenesis].

The INK4a-ARF locus, localized on 9p21, encodes two tumor suppressor proteins, p16INK4a and p14ARF, acting respectively through the CDK4-pRb and the p53 pathways. Familial melanoma (comprising between 8 and 12% of all melanoma cases) is a genodermatosis transmitted as an autosomal dominant trait, often associated with clinically atypical moles (AN). Germline mutations of p16INK4a are found in up to 20-30% of melanoma prone families. Mutated families often contain more than three family members affected and/or comprise at least one relative with multiple melanomas. Most of these mutations have been shown to affect p16INK4a protein function (i.e. CDK4 binding or pRB phosphorylation). Germline mutations of p16INK4a are also found in a lesser extend in sporadic multiple melanoma and in familial pancreatic cancer. The INK4a-ARF locus plays also an important role in skin carcinogenesis. P16INK4a UV induced mutations (CC:GG > TT:AA tandem transition or C:G > T:A transition at dipyrimidic site) are found in 12% of sporadic skin carcinomas, mainly in epidermoid tumors, and seem to occur independently of p53 mutations. Xeroderma pigmentosum (XP) is characterized by an inheritable DNA repair defect (involving the nucleotid excision repair (NER) system) predisposing to skin carcinomas. In skin tumors from (XP) patients, p16INK4a UV induced mutations occur more frequently, are often multiple, and significantly associated with the presence of p53 mutations. Such data, which could be related to the XP genetic instability and indicates a possible cooperative effect of inactivation of these pathways in the tumoral process of XP skin tumors.

Cyclin-Dependent Kinase Inhibitor p16↗
PubMedDate not supplied

Genetics of Aux/IAA and ARF action in plant growth and development.

Dramatic advances in our understanding of auxin signal-response pathways have been made in recent years. Much of this new knowledge has come through the study of mutants in Arabidopsis thaliana. Mutations have been identified in a wide variety of auxin-response components, including auxin transporters, protein kinases and phosphatases, components of a ubiquitin-proteosome pathway, and transcriptional regulators. This review focuses on mutations that affect auxin-modulated transcription factors, in particular those in the Aux/IAA and AUXIN RESPONSE FACTOR (ARF) genes. Mutants in members of these related gene families exhibit phenotypes that indicate both unique localized functions, as well as overlapping redundant functions, throughout plant development - from embryogenesis to flowering. Effects of specific mutations on Aux/IAA and ARF protein functions at the biochemical and physiological levels will be discussed. We will also discuss potential mechanisms for interactions between auxin and light response pathways that are suggested by these mutants.

Arabidopsis↗
PubMed1995

The effect of ArF-excimer laser irradiation of the human enamel surface on the bond strength of orthodontic appliances.

This study investigated enamel laser conditioning as an alternative to acid etching in bracket therapy. In preliminary experiments optimal laser parameters for achieving a bond strength of 6-10 N/mm2 were defined. Enamel surface morphology was assessed and the ablation depth was measured on serial enamel sections. Thirty human molars were exposed to 193 nm ArF-excimer laser radiation (energy density: 260 mJ/cm2) by single pulse application of 23 nanoseconds. Thirty molars were etched with phosphoric acid (37%) for 60 seconds. The brackets from the treated molars and 30 untreated molars were debonded vertically for tensile bond strength measurement. Roughened enamel surfaces were attained by 450 and 900 laser pulses with a mean ablation depth of 10.13 +/- 4.84 microns. After 1-10 laser pulses, the enamel surface appeared intact. The tensile bond strength was 6.63 +/- 2.18 N/mm2 in the laser-treated group (1 pulse), 8.75 +/- 3.61 N/mm2 in the acid-etched group, and 4.61 +/- 3.15 N/mm2 in the untreated group. We conclude a laser-selective ablation of the membranous enamel pellicle. Since the irradiated area can be adapted to bracket base and the enamel surface remains morphologically intact, pulsed ArF-excimer laser treatment seems to be superior to the acid etching technique.

Acid Etching, Dental↗