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PubMed2003

p19ARF-induced p53-independent apoptosis largely occurs through BAX.

Combined disruption of the ARF gene and the p53 gene causes mouse predisposition to tumors of a wider variety and at a higher frequency than disruption of the p53 gene, indicating that the ARF gene has p53-independent anti-tumor function in addition to p53-dependent function. Coincidentally with this notion, ectopic expression of the p19(ARF) induces apoptosis for wild-type mouse embryo fibroblasts which have been immortalized by introduction of the SV40 virus genome (SV40-MEFs). The protein expression levels of p53, p21(Cip1), and Bax were not upregulated by ectopic expression of p19(ARF) in SV40-MEFs, indicating that expression of p19(ARF) induced apoptosis through p53-independent pathways in this system. Ectopic expression of p19(ARF) induced prominent apoptosis even in SV40-Bak-/-MEFs. In contrast, expression of p19(ARF) induced only a very low grade of apoptosis in Bax-/- or Bax-/-/Bak-/-SV40-MEFs. Remarkable attenuation of p19(ARF)-induced apoptosis by disruption of the Bax gene thus leads to the conclusion that Bax plays a major role in p53-independent apoptosis induced by p19(ARF).

Animals↗
PubMed2005

PKCalpha is involved in phorbol ester TPA-mediated stabilization of p14ARF.

We generated A21-13 cells expressing p14(ARF) in the presence of doxycycline in order to examine the stability of p14(ARF) protein. The effects of proteasome inhibitor MG132 on p14(ARF) protein stabilization were detectable using our experimental procedure. Introduction of mutant p53 did not affect MG132-mediated p14(ARF) protein stabilization. We found that phorbol ester TPA (12-o-tetradecanoyl-phorbol 13-acetate) stabilized p14(ARF) protein and that p53 status had no effect on TPA-mediated stabilization. TPA-mediated stabilization was abolished by staurosporine but not by lovastatin or U0126. We further investigated which isoforms of PKC were involved in TPA-mediated p14(ARF) stabilization using short-interference RNA. Knockdown of PKCalpha, but not PKCdelta, attenuated TPA-mediated p14(ARF) stabilization. These findings suggest that PKCalpha is involved in TPA-mediated stabilization of p14(ARF) protein, and this effect of TPA was not affected by the Ras/MAPK pathway or p53 status. Our results are indicative of a novel role of PKC in p14(ARF) protein stability.

Butadienes↗
PubMed2005

The challenge of acute rheumatic fever diagnosis in a high-incidence population: a prospective study and proposed guidelines for diagnosis in Australia's Northern Territory.

BACKGROUND: Accurate diagnosis of acute rheumatic fever (ARF) remains problematic in high-incidence settings and especially in the Aboriginal population of Australia's Northern Territory. Previous investigators have demonstrated that strict application of the 1992 Updated Jones Criteria results in under-diagnosis. This study's objectives were to review use of the Jones Criteria (1992 Update) in diagnosing ARF in Australian Aboriginal patients presenting with suspected rheumatic fever, and formulate a locally relevant algorithm to improve diagnosis. METHODS: Patients presenting to Royal Darwin Hospital with suspected ARF were prospectively assessed during a 15-month period. Demographic information, clinical history, examination, laboratory and echocardiographic findings were documented in order to determine whether the Jones Criteria were fulfilled, and to identify alternative diagnoses. The hospital discharge diagnosis was recorded and patients were followed up 18-33 months later. RESULTS: Out of 35 patients with suspected ARF, all were Aboriginal Australians, 17 (49%) had a discharge diagnosis of definite ARF, 7 (20%) had definite non-rheumatic fever diagnoses (disseminated gonococcal infection, systemic lupus erythematosis, buttock abscess and other febrile illnesses in children with cardiac murmur due to previously undiagnosed RHD). The remaining 11 (31%) posed diagnostic difficulties because of mild symptoms that failed to fulfil Jones Criteria (attracting diagnoses such as 'unexplained arthralgia') or atypical features such as older age. Two patients whose illness initially failed to fulfil the Jones Criteria, who were neither diagnosed with ARF nor commenced on secondary benzathine penicillin prophylaxis, were found on follow-up to have definite and probable ARF, respectively. At least 29% (8/28) of patients without prior recognised ARF/RHD had echocardiographic evidence of established RHD, indicating that previous episodes were missed. CONCLUSIONS: Individual mild episodes of ARF may be overlooked, with patients missing out on the timely institution of secondary prophylaxis. The Jones Criteria should be supplemented by active exclusion of differential diagnoses and vigilant follow-up including echocardiography. 'Probable' and 'possible ARF' should be recognised as diagnostic categories applying to patients not fulfilling the Jones Criteria but who nevertheless should be offered prophylactic penicillin at least until further follow-up. A set of diagnostic guidelines is proposed.

Acute Disease↗
PubMed2004

Profiles of the 2 INK4a gene products, p16 and p14ARF, in human reference urothelium and bladder carcinomas, according to pRb and p53 protein status.

The INK4a/ARF locus encodes 2 cell cycle regulatory proteins: p16 and p14(ARF). P16 inhibits the activities of cdks, which maintain the retinoblastoma protein (pRb) in its active hypophosphorylated state. P14(ARF) blocks MDM2-induced p53 degradation and transactivational silencing. In this study, we investigated the expression of p16 and p14(ARF) in reference human urothelium and in 51 urothelial carcinomas (UCs) of all stages and grades, by reverse transcription-polymerase chain reaction (RT-PCR). Patterns of p14(ARF) and p16 expression were compared with each other and then with patterns of p53 and pRb protein expression, respectively, as determined by immunohistochemistry. P14(ARF) and p16 mRNAs were present at low levels or were undetectable in reference urothelia and in most superficial tumors, whereas they were present at high levels in a subset of tumors of advanced stage and high grade. The expression profiles of these 2 mRNAs were correlated in all but 4 cases, indicating that the 2 INK4a products may have nonredundant functions. Forty-six of the 51 tumors (90%) presented changes to or a lack of activation of the p14(ARF)-p53 pathway and were p53 positive (n = 10), p14(ARF) negative (n = 23), or both p53 positive and p14(ARF) negative (n = 13), suggesting that these 2 components of the pathway may be altered or nonactivated. Markedly high levels of p16 mRNA (n = 5) were associated with the absence of pRb expression, with the exception of 1 case in which the p16 gene contained a deletion mutation. A lack of p16 mRNA or low levels of this mRNA were associated with pRb detection in all but 1 case. In invasive UCs, the p16-pRb pathway, the p14(ARF)-p53 pathway, or in many cases both pathways were altered or not activated, demonstrating the involvement of these pathways in invasive bladder tumorigenesis.

Carcinoma↗
PubMed2006

Clinical and laboratory predictors of oliguric renal failure in haemorrhagic fever with renal syndrome caused by Hantaan virus.

OBJECTIVE: Haemorrhagic fever with renal syndrome (HFRS), caused by hantavirus infection, develops into acute renal failure (ARF) of variable degrees of severity. We investigated the early predictive markers for oliguric ARF in HFRS patients. METHODS: A retrospective cohort study was performed of 61 patients with HFRS between 2000 and 2004. These patients were categorized into either oliguric or non-oliguric ARF groups according to their urine output (<400 ml/24 h). The clinical characteristics were compared between the two groups. RESULTS: Of the 61 patients, 24 (39.3%) were classified as oliguric ARF and 37 (60.7%) as non-oliguric ARF. The peak serum Cr was 10.8 (IQR 9.1-12.4) mg/dl in oliguric ARF and 4.4 (IQR 3.1-6.0) mg/dl in non-oliguric ARF (p<0.001). The risk for developing oliguric ARF significantly increased in the cases with leukocyte count (> or =14 x 10(9)/L, aOR 2.2, 95% CI 1.0-4.9; p=0.039), elevated aspartate aminotransferase (> or =110 U/L, aOR 11.0, 95% CI 2.1-57.9; p=0.005) and the presence of microscopic haematuria (> or =5/HPF, aOR 9.2, 95% CI 1.4-60.3; p=0.021) at the time of admission. CONCLUSION: The leukocyte count, level of aspartate aminotransferase and microscopic haematuria at admission would be useful to predict for the subsequent development of oliguric ARF in HFRS.

Acute Kidney Injury↗
PubMed2004

Assessment of acute renal failure patients treated in our nephrology clinic between 1996 and 2002.

Acute renal failure (ARF) is a cause of high morbidity and mortality associated with long hospital stay, and expensive treatment. The initial approach to patients with ARF should be focused on preventing future injury to the kidney. Two hundred eighty-three ARF patients, treated from January 1996 to June 2002, were retrospectively investigated for their etiology, clinic features, and laboratory characteristics, as well as treatment results and mortality rate. The mean age was 52.3 +/- 18.7 years. Patients with hospital-acquired ARF comprised 38.8% of the sample. Renal causes (60%) were responsible for most ARF patients. They were medical (63.95%), surgical (23.67%), and obstetric (12.4%) causes. Twenty-five percent of patients with ARF had multiple etiologies. Hemolysis elevated liver enzymes low platelets (HELLP) syndrome was seen in the most of the obstetric-related ARF cases. Signs of hypervolemia were present in approximately 50% of the cases. Oliguric patients comprised 59.7% of the sample, and the mean time to oliguria was 5.2 +/- 4.1 days. The necessity of dialysis was greater in oliguric patients (42.6%) and the ratio of complete/partial improvement (82.2%) was greater among non-oligoanuric patients. However, there was no significant difference between mortality rates. Irreversible renal insufficiency did not develop in the non-oliguric cases. Also, 7.4% of ARF patients died, with the main causes being infection (31.8%) and cardiovascular events (27.2%). Medical problems are important in the etiology of ARF as well as obstetric cases. The mortality rate was low in our cases, a situation that may be explained by medical causes being of importance in the etiology. We are of the opinion that early referral of patients to a nephrologist and following treatment in the nephrology clinic may positively affect the outcome.

Acute Kidney Injury↗
PubMed2000

INK4a/ARF locus alterations in human non-Hodgkin's lymphomas mainly occur in tumors with wild-type p53 gene.

INK4a/ARF locus codes for two different proteins, p16(INK4a) and p14(ARF), involved in cell cycle regulation. p14(ARF) is considered an upstream regulator of p53 function. To determine the role of these genes in the pathogenesis of human non-Hodgkin's lymphomas we have analyzed exon 1beta, 1alpha, and 2 of the INK4a/ARF locus and p53 gene aberrations in 97 tumors previously characterized for p16(INK4a) alterations. p53 alterations were detected in four of 51 (8%) indolent lymphomas but in 15 of 46 (33%) aggressive tumors. Inactivation of p14(ARF) was always associated with p16(INK4a) alterations. Exon 1beta was concomitantly deleted with exon 1alpha and 2 in eight tumors. One additional lymphoblastic lymphoma showed deletion of exon 1alpha and 2 but retained exon 1beta. No mutations were detected in exon 1alpha and 1beta in any case. Two of the three mutations detected in exon 2 caused a nonsense mutation in the p16(INK4a) reading frame and a missense mutation in the ARF reading frame involving the nucleolar transport domain of the protein. The third mutation was a missense mutation in the p16(INK4a) reading frame, but it was outside the coding region of p14(ARF). Aggressive lymphomas with p14(ARF) inactivation and p53 wild type showed a significantly lower p53 protein expression than tumors with no alteration in any of these genes. In this series of tumors, inactivation of the INK4a/ARF locus mainly occurred in tumors with a wild-type p53 gene because only two lymphomas showed simultaneous aberrations in these genes. Tumors with concomitant alterations of p16(INK4a) and p14(ARF)/p53 genes seem to exhibit a worse clinical behavior than lymphomas with no alterations or isolated inactivation of any of these genes. These findings indicate that p14(ARF) genetic alterations occur in a subset of aggressive NHLs, but they are always associated with p16(INK4a) aberrations. Concomitant disruption of p16(INK4a) and p14(ARF)/p53 regulatory pathways may have a cooperative effect in the progression of these tumors.

Carrier Proteins↗
PubMed2005

Preparation of myristoylated Arf1 and Arf6.

Arf proteins are members of the Arf family of small Ras-like GTP binding proteins. Six Arfs, grouped into three classes, have been identified in mammalian cells and three members have been identified in yeasts. Arf1 and Arf6, more extensively studied than other Arfs, have been found to affect membrane traffic and actin remodeling. A structural feature that distinguishes Arfs from other Ras superfamily members is an N-terminal alpha-helix, extending from the basic G-protein fold, which is cotranslationally myristoylated. Both the helix and the myristate affect biochemical properties of Arfs, including nucleotide exchange, membrane association, and interaction with some effector proteins. Preparation of myristoylated Arf for in vitro studies of Arf function requires consideration of both the reaction yielding myristoylated protein and the properties of the modified Arfs. Here, we describe methods that yield homogeneous preparations of myristoylated Arf1 and Arf6.

ADP-Ribosylation Factor 1↗
PubMed1985

[Acute renal failure after extracorporeal circulation with aortic counterpulsation in surgically treated patients].

In a series of 604 adults operated on for cardiac surgery with cardiopulmonary bypass (CPB), 21 (3.5%) underwent circulatory assistance by intra-aortic balloon pump (IABP); in 5 of them (24%), acute renal failure (ARF) was observed. ARF occurred in only 26 (4.4%) of the other patients who did not require IABP. Evolution of ARF and its factors were therefore investigated in those patients having received IABP. ARF was defined as serum blood urea nitrogen (BUN) greater than or equal to 16 mmol X 1(-1), urinary urea/BUN less than 10, creatinine clearance less than 40 ml X min-1 X 1.73 m-2. Some perioperative features were compared between patients with postoperative ARF and those without ARF. ARF occurred in the 5 patients with IABP during, or immediately after, weaning from IABP. ARF was more frequent in patients operated on for mechanical complications of myocardial infarction with a significant more severe haemodynamic status. They had significantly longer CPB and aortic clamping times. The prognosis depended on the cardiac failure and not on the ARF. In patients with mechanical complications of infarction, early IABP seemed to be the predominant preventive measure. Other therapeutic implications are suggested, particularly the use of dopamine (1 to 3 micrograms X kg-1 X min-1) because of its renal vasodilating action which can contribute to the maintenance of urinary flow.

Acute Kidney Injury↗
PubMed1991

Guanine nucleotide dependent formation of a complex between choleragen (cholera toxin) a subunit and bovine brain ADP-ribosylation factor.

Cholera toxin activates adenylyl cyclase by catalyzing the ADP-ribosylation of Gs alpha, the stimulatory guanine nucleotide binding protein of the cyclase system. This toxin-catalyzed reaction, as well as the ADP-ribosylation of guanidino compounds and auto-ADP-ribosylation of the toxin A1 protein (CTA1), is stimulated, in the presence of GTP (or GTP analogue), by 19-21-kDa proteins, termed ADP-ribosylation factors or ARFs. These proteins directly activate CTA1 in a reaction enhanced by sodium dodecyl sulfate (SDS) or dimyristoylphosphatidylcholine (DMPC)/cholate. To determine whether ARF stimulation of ADP-ribosylation is associated with formation of a toxin-ARF complex, these proteins were incubated with guanine nucleotides and/or detergents and then subjected to gel permeation chromatography. An active ARF-toxin complex was observed in the presence of SDS and GTP gamma S [guanosine 5'-O-(3-thiotriphosphate)] but not GDP beta S [guanosine 5'-O-(2-thiodiphosphate)]. Only a fraction of the ARF was capable of complex formation. The substrate specificities of complexed and noncomplexed CTA differed; complexed CTA exhibited markedly enhanced auto-ADP-ribosylation. In the presence of GTP gamma S and DMPC/cholate, an ARF-CTA complex was not detected. A GTP gamma S-dependent ARF aggregate was observed, however, exhibiting a different substrate specificity from monomeric ARF. These studies support the hypothesis that in the presence of guanine nucleotide and either SDS or DMPC/cholate, ARF and toxin exist as multiple species which exhibit different substrate specificities.

ADP-Ribosylation Factors↗
PubMed1992

Decreased systemic clearance of diltiazem with increased hepatic metabolism in rats with uranyl nitrate-induced acute renal failure.

The effect of uranyl nitrate (UN)-induced acute renal failure (ARF) on the pharmacokinetics of diltiazem (DTZ) was examined in rats through in vitro and in vivo studies. In vitro homogenate studies demonstrated that DTZ was metabolized to deacetyl diltiazem (DAD) predominantly in the liver. Metabolism in the small intestine, kidney, or blood pool was negligible compared with that in the liver. UN-induced ARF (UN-ARF) increased the in vitro hepatic clearance (CLvit) of DTZ 1.4-fold. In vivo pharmacokinetic studies following intravenous (iv) and portal venous (pv) administration revealed that UN-ARF increased the intrinsic clearance (CLi) of DTZ from 243.0 to 414.5 ml/min/kg but decreased its total plasma clearance (CLt) from 90.3 to 64.3 ml/min/kg. The increase in CLi was consistent with the increase in CLvit of the liver. The in vitro plasma free fraction of DTZ (fp) was decreased from 0.25 to 0.14 by UN-ARF, but the in vitro blood/plasma partition of DTZ (Rb) remained constant at unity. From the CLi and fp changes, the plasma intrinsic clearance for unbound DTZ (CLi') was calculated to be increased 2.7-fold, from 1104.5 to 2960.7 ml/min/kg, by UN-ARF. The fp decrease was also reflected in the steady-state distribution volume (Vdss) of DTZ, which was decreased significantly from 3595.5 to 2528.3 ml/kg. The absolute bioavailability of pv DTZ (Fpv) was decreased by UN-ARF from 37.5 to 15.5% but was still much higher than the reported oral bioavailability (6%), indicating poor absorption of DTZ from the GI tract. From the calculation based on a well-stirred pharmacokinetic model, DTZ was found to increase the hepatic blood flow (HBF) of the control rats more than twofold at doses of 3 mg/kg (iv) or 10 mg/kg (pv), possibly due to the vasodilating effect of DTZ. However, the effect of DTZ on HBF was not present in the UN-ARF rats. It is not clear at present whether this could be attributed to vasoconstricting effects of UN-ARF or blockade of the vasodilating effect of DTZ.

Acute Kidney Injury↗
PubMed1994

Responses to hemorrhagic arterial pressure reduction in different ischemic renal failure models.

Renal blood flow (RBF) autoregulation has been found to be impaired in both norepinephrine (NE) and renal artery clamp (RAC) rat ischemic acute renal failure models. However, the decline in RBF at the normal lower limit of autoregulation is greater in NE-ARF. The present study was designed to determine if this difference in autoregulatory profiles has potential functional and morphologic significance. After demonstrating a fall in RBF to renal perfusion pressure reduction to 90 mm Hg that was twofold more in one week NE- than RAC-ARF (p < 0.001), separate rats of both ischemic ARF types with nearly identical levels of azotemia and glomerular filtration rate reduction and sham-ARF rats were subjected to four-hour controlled reduction in mean arterial pressure to 90 by transient phlebotomy at one week. On day 9, two days after mean arterial pressure reduction, blood urea nitrogen (BUN), serum creatinine (SCr) and creatinine clearance (CCr) showed continued improvement in RAC-ARF, but there were significant increases in BUN (46 +/- 22 to 72 +/- 10 mg/dl) and SCr (1.2 +/- 0.2 to 1.5 +/- 0.2 mg/dl) and a decline in CCr (0.434 +/- 0.127 to 0.334 +/- 0.079 ml/min) in the NE-ARF group (all P < 0.02). The mean sum of scores of morphologic indices of ARF was higher in NE- than RAC-ARF kidneys of rats sacrificed on day 9 but interstitial edema was the only individual index that was worse in NE-ARF.(ABSTRACT TRUNCATED AT 250 WORDS)

Acute Kidney Injury↗
PubMed2006

Biomarker and drug-target discovery using proteomics in a new rat model of sepsis-induced acute renal failure.

Sepsis is one of the common causes of acute renal failure (ARF). The objective of this study was to identify new biomarkers and therapeutic targets. We present a new rat model of sepsis-induced ARF based on cecal ligation and puncture (CLP). We used this model to find urinary proteins which may be potential biomarkers and/or drug targets. Aged rats were treated with fluids and antibiotics after CLP. Urinary proteins from septic rats without ARF and urinary proteins from septic rats with ARF were compared by difference in-gel electrophoresis (DIGE). CLP surgery elevated interleukin (IL)-6 and IL-10 serum cytokines and blood nitrite compared with sham-operated rats. However, there was a range of serum creatinine values at 24 h (0.4-2.3 mg/dl) and only 24% developed ARF. Histology confirmed renal injury in these rats. Forty-nine percent of rats did not develop ARF. Rats without ARF also had less liver injury. The mortality rate at 24 h was 27% but was increased by housing the post-surgery rats in metabolic cages. Creatinine clearance and urine output 2-8 h after CLP was significantly reduced in rats which died within 24 h. Using DIGE we identified changes in a number of urinary proteins including albumin, brush-border enzymes (e.g., meprin-1-alpha) and serine protease inhibitors. The meprin-1-alpha inhibitor actinonin prevented ARF in aged mice. In summary, we describe a new rat model of sepsis-induced ARF which has a heterogeneous response similar to humans. This model allowed us to use DIGE to find changes in urinary proteins and this approach identified a potential biomarker and drug target - meprin-1-alpha.

Acute Kidney Injury↗
PubMed2006

Mortality after acute renal failure: models for prognostic stratification and risk adjustment.

To adjust adequately for comorbidity and severity of illness in quality improvement efforts and prospective clinical trials, predictors of death after acute renal failure (ARF) must be accurately identified. Most epidemiological studies of ARF in the critically ill have been based at single centers, or have examined exposures at single time points using discrete outcomes (e.g., in-hospital mortality). We analyzed data from the Program to Improve Care in Acute Renal Disease (PICARD), a multi-center observational study of ARF. We determined correlates of mortality in 618 patients with ARF in intensive care units using three distinct analytic approaches. The predictive power of models using information obtained on the day of ARF diagnosis was extremely low. At the time of consultation, advanced age, oliguria, hepatic failure, respiratory failure, sepsis, and thrombocytopenia were associated with mortality. Upon initiation of dialysis for ARF, advanced age, hepatic failure, respiratory failure, sepsis, and thrombocytopenia were associated with mortality; higher blood urea nitrogen and lower serum creatinine were also associated with mortality in logistic regression models. Models incorporating time-varying covariates enhanced predictive power by reducing misclassification and incorporating day-to-day changes in extra-renal organ system failure and the provision of dialysis during the course of ARF. Using data from the PICARD multi-center cohort study of ARF in critically ill patients, we developed several predictive models for prognostic stratification and risk-adjustment. By incorporating exposures over time, the discriminatory power of predictive models in ARF can be significantly improved.

APACHE↗
PubMed2002

Methylation, expression, and mutation analysis of the cell cycle control genes in human brain tumors.

Methylation status of the p15(INK4B), p16(INK4A), p14(ARF) and retinoblastoma (RB) genes was studied using methylation specific polymerase chain reaction (MSP) in 85 human brain tumors of various subtypes and four normal brain samples. These genes play an important role in the control of the cell cycle. Twenty-four out of 85 cases (28%) had at least one of these genes methylated. The frequency of p14(ARF) methylation was 15 out of 85 (18%) cases, and the expression of p14(ARF) in methylated gliomas was significantly lower than in unmethylated gliomas. The incidence of methylation of p15(INK4B), p16(INK4A) and RB gene was 4%, 7%, and 4%, respectively. Samples with p14(ARF) methylation did not have p16(INK4A) methylation even though both genes physically overlap. None of the target genes was methylated in the normal brain samples. In addition, the p53 gene was mutated in 19 out of 85 (22%) samples as determined by single strand conformation polymorphism (SSCP) analysis and DNA sequencing. Thirty out of 85 (35%) brain tumors had either a p53 mutation or methylation of p14(ARF). Also, the p14(ARF) expression in p53 wild-type gliomas was lower than levels in p53 mutated gliomas. This finding is consistent with wild-type p53 being able to autoregulate its levels by down-regulating expression of p14(ARF). In summary, inactivation of the apoptosis pathway that included the p14(ARF) and p53 genes by hypermethylation and mutation, respectively, occurred frequently in human brain tumors. Down-regulation of p14(ARF) in gliomas was associated with hypermethylation of its promoter and the presence of a wild-type p53 in these samples.

Base Sequence↗
PubMed2003

P19ARF inhibits the functions of the HPV16 E7 oncoprotein.

The E7 oncoprotein encoded by high-risk types of human papillomavirus (HPV) plays a significant role in the development of HPV-related cancers. E7 is a potent stimulator of S phase and host DNA replication. These functions of E7 are linked to the deregulation of the Rb family of proteins. For example, E7 binds and induces proteolysis of Rb through the ubiquitin-proteasome pathway. Despite advances in our understanding of E7, reagents that inhibit E7 with promise in therapy have not been developed or identified. Here, we provide evidence that the tumor suppressor ARF can inhibit E7. We show that the expression of ARF causes a relocalization of E7 from the nucleoplasm to the nucleolus. Two distinct regions in ARF overlapping with the MDM2-binding sites are necessary for the relocalization of E7. Furthermore, we show that ARF blocks the proteolysis of Rb induced by E7. In addition, ARF expression inhibits DNA replication induced by E7. Although it is not known whether the endogenous ARF, which is expressed at a low level, interferes with E7, our results suggest that ARF is an effective inhibitor of E7. We speculate that ARF or an ARF-derived molecule might have a significant impact in therapy against HPV-related tumors.

Cell Nucleolus↗
PubMed1997

Evidence that a low-molecular-mass GTP-binding protein is required for store-activated Ca2+ inflow in hepatocytes.

The roles of a monomeric GTP-binding regulatory protein in the activation of store-activated plasma membrane Ca2+ channels and in the release of Ca2+ from the smooth endoplasmic reticulum (SER) in rat liver parenchymal cells were investigated with the use of freshly isolated rat hepatocytes and rat liver microsomes. A low concentration (approx. 130 microM intracellular) of guanosine 5'-[gamma-thio]triphosphate (GTP[S]) activated Ca2+ inflow in intact hepatocytes in the absence of an agonist, whereas a high concentration (approx. 530 microM intracellular) of GTP-S- or guanosine 5'-[betagamma-imido]triphosphate (p[NH]ppG) inhibited the Ca2+ inflow induced by inhibitors of the activity of the endoplasmic-reticulum Ca2+-ATPase (SERCA) and by vasopressin. GTP (530 microM) prevented the inhibition of Ca2+ inflow by GTP-S- and p[NH]ppG. Brefeldin A and the peptide human Arf-1-(2-17), which inhibit many functions of ADP ribosylation factor (Arf) proteins, inhibited the Ca2+ inflow induced by SERCA inhibitors and vasopressin, and altered the profile of Ca2+ release from the SER. These effects were observed at concentrations of Brefeldin A and Arf-1-(2-17) comparable with those that inhibit the functions of Arf proteins in other systems. Succinylated Arf-1-(2-17) had a negligible effect on Ca2+ inflow. GTP[S] and Arf-1-(2-17) completely inhibited the synergistic action of GTP and Ins(1,4,5)P3 in releasing 45Ca2+ from rat liver microsomes loaded with 45Ca2+. AlF4(-) (under conditions expected to activate trimeric G-proteins) and succinylated Arf-1-(2-17) had no effect on GTP/Ins(1,4,5))3-induced 45Ca2+ release, and a mastoparan analogue caused partial inhibition. Arf-1-(2-17) did not inhibit 45Ca2+ release induced by either thapsigargin or ionomycin. It is concluded that a low-molecular-mass G-protein, most probably a member of the Arf protein family, is required for store-activated Ca2+ inflow in rat hepatocytes. The idea that the role of this G-protein is to maintain a region of the SER in the correct intracellular location is discussed briefly.

ADP-Ribosylation Factors↗
PubMed1999

Dimerization and DNA binding of auxin response factors.

Auxin response factors (ARFs) are transcription factors that bind with specificity to TGTCTC auxin response elements (AuxREs) found in promoters of primary/early auxin response genes. ARFs are encoded by a multi-gene family, consisting of more than 10 genes. Ten ARFs have been analyzed by Northern analysis and were found to be expressed in all major plant organs and suspension culture cells of Arabidopsis. The predicted amino acid sequences indicate that the 10 ARFs contain a novel amino-terminal DNA binding domain and a carboxyl-terminal dimerization domain, with the exception of ARF3 which lacks this dimerization domain. All ARFs tested bind with specificity to the TGTCTC AuxRE, but there are subtle variations in the sequence requirements at positions 5 (T) and 6 (C) of the AuxRE. While the amino-terminal domain of about 350 amino acids is sufficient for binding ARF1 to TGTCTC AuxREs, this domain is not sufficient for the binding of some other ARFs to palindromic AuxREs. Our results suggest that ARFs must form dimers on palindromic TGTCTC AuxREs to bind stably, and this dimerization may be facilitated by conserved motifs found in ARF carboxyl-terminal domains. Dimerization in at least some cases may dictate which ARF(s) are targeted to AuxREs.

Amino Acid Sequence↗