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R Hickey

Publications and source records attributed to R Hickey.

At least 37 records · Page 2Linked to original sources
PubMed1997

The biochemical status of the DNA synthesome can distinguish between permanent and temporary cell growth arrest.

We previously identified and characterized the human leukemia (HL-60) cell DNA synthetic machinery as a multiprotein form of DNA polymerase, which was designated the DNA synthesome. This multiprotein replication complex contains DNA polymerases alpha and delta, primase, replication factor C, replication protein A, helicase, poly(ADPribose) polymerase, proliferating cell nuclear antigen, DNA ligase I, and topoisomerases I and II. Recently, the HeLa cell-derived DNA synthesome was identified as a discrete high molecular weight protein band in native polyacrylamide gels. Here, we report our findings regarding the change in the organizational status of the DNA synthesome when HL-60 cells undergo either terminal differentiation or temporary G1 growth arrest. We observed that the HL-60 cell DNA synthesome also migrates as a discrete high molecular weight protein band in nondenaturing polyacrylamide gels. This high molecular weight protein band was present in nuclei derived from both actively cycling cells and aphidicolin-arrested cells but was absent in TPA-induced terminally differentiated cells. We also found that DNA polymerase delta, replication factor C, and proliferating cell nuclear antigen are absent in cells that are induced to differentiate in response to 12-O-tetradecanoyl phorbol-13-acetate treatment but are present in actively cycling cells. The level of replication protein A in differentiated cells was similar to that of cycling cells, whereas the level of annexin I, a cytoskeleton protein, is higher in differentiated cells than it is in actively cycling cells. We conclude that the DNA synthesome remains integrated and inactive in temporarily growth-arrested cells but is disassembled in differentiated cells. Furthermore, we conclude that disassembly of the organized replication complex is a specific cellular event in the process of permanent cell cycle exit and that the process leading to disassembly may be regulated, in part, at the level of gene transcription.

Aphidicolin↗
PubMed1996

The expression of poly(ADP-ribose) polymerase during differentiation-linked DNA replication reveals that it is a component of the multiprotein DNA replication complex.

3T3-L1 preadipocytes have been shown to exhibit a transient increase in poly(ADP-ribose) polymerase (PARP) protein and activity, as well as an association of PARP with DNA polymerase alpha, within 12-24 h of exposure to inducers of differentiation, whereas 3T3-L1 cells expressing PARP antisense RNA showed no increase in PARP and are unable to complete the round of DNA replication required for differentiation into adipocytes. The role of PARP in differentiation-linked DNA replication has now been further clarified at both the cellular and enzymological levels. Flow cytometric analysis revealed that control 3T3-L1 cells progressed through one round of DNA replication prior to the onset of terminal differentiation, whereas cells expressing PARP antisense RNA were blocked at the G0/G1 phase of the cell cycle. Confocal microscope image analysis of control S phase cells demonstrated that PARP was localized within distinct intranuclear granular foci associated with DNA replication centers. On the basis of these results, purified replicative complexes from other cell types that had been characterized for their ability to catalyze viral DNA replication in vitro were analyzed for the presence of PARP. PARP exclusively copurified through a series of centrifugation and chromatography steps with core proteins of an 18-21S multiprotein replication complex (MRC) from human HeLa cells, as well as with the corresponding mouse MRC from FM3A cells. The MRC were shown to contain DNA polymerases alpha and delta, DNA primase, DNA helicase, DNA ligase, and topoisomerases I and II, as well as accessory proteins such as PCNA, RF-C, and RP-A. Finally, immunoblot analysis of MRCs from both cell types with monoclonal antibodies to poly (ADP-ribose) revealed the presence of approximately 15 poly(ADP-ribosyl)ated proteins, some of which were further confirmed to be DNA polymerase alpha, DNA topoisomerase I, and PCNA by immunoprecipitation experiments. These results suggest that PARP may play a regulatory role within the replicative apparatus as a molecular nick sensor controlling the progression of the replication fork or modulates component replicative enzymes or factors in the complex by directly associating with them or by catalyzing their poly(ADP-ribosyl)ation.

3T3 Cells↗
PubMed1996

A novel in vitro model system for studying the action of ara-C.

The antimetabolite 1-beta-D-arabinofuranosyl-cytosine (ara-C) has proven to be one of the most effective agents available for the treatment of acute leukemia. While ara-C has been implicated as a potent inhibitor of mammalian cell DNA replication, the specific mechanism by which ara-C kills cells is not known. In this report we describe the development of an in vitro model system to study the molecular mechanism of ara-CMP incorporation into DNA. This model system makes use of a recently described human cell multiprotein DNA replication complex (MRC) that is competent to replicate DNA in vitro. The MRC can successfully incorporate ara-CMP into replicating DNA at internucleotide positions. These results are similar to those described for studies using intact cells. This MRC-driven in vitro replication system may therefore serve as a powerful model for the study of anticancer agents that directly affect human cell DNA synthesis.

Antimetabolites, Antineoplastic↗
PubMed1994

A 17S multiprotein form of murine cell DNA polymerase mediates polyomavirus DNA replication in vitro.

We have identified and purified a multiprotein form of DNA polymerase from the murine mammary carcinoma cell line (FM3A) using a series of centrifugation, polyethylene glycol precipitation, and ion-exchange chromatography steps. Proteins and enzymatic activities associated with this mouse cell multiprotein form of DNA polymerase include the DNA polymerases alpha and delta, DNA primase, proliferating cell nuclear antigen (PCNA), DNA ligase I, DNA helicase, and DNA topoisomerases I and II. The sedimentation coefficient of the multiprotein form of DNA polymerase is 17S, as determined by sucrose density gradient analysis. The integrity of the murine cell multiprotein form of DNA polymerase is maintained after treatment with detergents, salt, RNase, DNase, and after chromatography on DE52-cellulose, suggesting that the association of the proteins with one another is independent of nonspecific interaction with other cellular macromolecular components. Most importantly, we have demonstrated that this complex of proteins is fully competent to replicate polyomavirus DNA in vitro. This result implies that all of the cellular activities required for large T-antigen dependent in vitro polyomavirus DNA synthesis are present within the isolated 17S multiprotein form of the mouse cell DNA replication activities. A model is proposed to represent the mammalian Multiprotein DNA Replication Complex (MRC) based on the fractionation and chromatographic profiles of the individual proteins found to co-purify with the complex.

Animals↗
PubMed1994

Positive regulatory elements (HF-1a and HF-1b) and a novel negative regulatory element (HF-3) mediate ventricular muscle-specific expression of myosin light-chain 2-luciferase fusion genes in transgenic mice.

The cardiac myosin light-chain 2v (MLC-2v) gene has served as a model system to identify the pathways which restrict the expression of cardiac muscle genes to particular chambers of the heart during cardiogenesis. To identify the critical cis regulatory elements which mediate ventricular chamber-specific expression of the MLC-2v gene in the in vivo context, a series of transgenic mice which harbor mutations in putative MLC-2 cis regulatory elements in a 250-bp MLC-2-luciferase fusion gene which is expressed in a ventricular chamber-specific fashion in transgenic mice were generated. These studies demonstrate that both components of HF-1 (HF-1a and HF-1b/MEF-2) are required to maintain ventricular chamber-specific expression and function as positive regulatory elements. Mutations in another conserved element (HF-2) are without statistically significant effect on ventricular chamber expression. Transgenics harboring mutations in the E-box site also displayed significant upregulation of reporter activity in the soleus, gastrocnemius, and uterus, with a borderline effect on expression in liver. Mutations in another conserved element (HF-3) result in a marked (> 75-fold) upregulation of the luciferase reporter activity in the soleus muscle of multiple independent or transgenic founders. Since the HF-3 mutations appeared to have only a marginal effect on luciferase reporter activity in liver tissue, HF-3 appears to function as a novel negative regulatory element to primarily suppress expression in muscle tissues. Thus, a combination of positive (HF-1a/HF-1b) and negative (E-box and HF-3) regulatory elements appear to be required to maintain ventricular chamber-specific expression in the in vivo context.

Animals↗
PubMed1993

Prevalence of urinary tract infection in febrile infants.

Urinary tract infection (UTI), a relatively common cause of fever in infancy, usually consists of pyelonephritis and may cause permanent renal damage. This study assessed (1) the prevalence of UTI in febrile infants (temperature > or = 38.3 degrees C) with differing demographic and clinical characteristics and (2) the usefulness of urinalysis in diagnosing UTI. We diagnosed UTI in 50 (5.3%) of 945 febrile infants if we found > or = 10,000 colony-forming units of a single pathogen per milliliter in a urine specimen obtained by catheterization. Prevalences were similar in (1) infants aged < or = 2 months undergoing examination for sepsis (4.6%), (2) infants aged > 2 months in whom UTI was suspected, usually because no source of fever was apparent (5.9%), and (3) infants with no suspected UTI, most of whom had other illnesses (5.1%). Female and white infants had significantly more UTIs, respectively, than male and black infants. In all, 17% of white female infants with temperature > or = 39 degrees C had UTI, significantly more (p < 0.05) than any other grouping of infants by sex, race, and temperature. Febrile infants with no apparent source of fever were twice as likely to have UTI (7.5%) as those with a possible source of fever such as otitis media (3.5%) (p = 0.02). Only 1 (1.6%) of 62 subjects with an unequivocal source of fever, such as meningitis, had UTI. As indicators of UTI, pyuria and bacteriuria had sensitivities of 54% and 86% and specificities of 96% and 63%, respectively. In infants with fever, clinicians should consider UTI a potential source and consider a urine culture as part of the diagnostic evaluation.

Bacteria↗
PubMed1993

Antihelicase action of DNA-binding anticancer agents: relationship to guanosine-cytidine intercalator binding.

DNA-binding antibiotics such as intercalators, narrow groove binders, and other substances modify duplex DNA, making it an altered substrate for DNA helicases. The intercalators daunorubicin, actinomycin D, echinomycin, and elsamicin, the narrow groove binders distamycin and mithramycin, and the plant toxin teniposide, each representing a different chemical class, block SV40 large T antigen DNA helicase action with IC50 values ranging from 4 x 10(-8) to 2 x 10(-6) M. A partially purified human HeLa cell DNA helicase is also potently blocked by daunorubicin, distamycin, and teniposide. Because eukaryotic cells contain helicases of varying abundance, specificity, and type, this site of action for DNA-binding antibiotics may help explain antibiotic potency and specificity for DNA or RNA inhibition. The antihelicase effect of the antibiotic-double-stranded DNA complex may be central to the anticancer activities of these substances. An additional interesting correlation is the antihelicase action of DNA-intercalating antibiotics and their DNA-binding preference for G-C base pair sites. The G-C base pair binding preference of the intercalating antibiotics may result from evolutionary selection because of the higher G-C binding stability, compared with A-T binding stability. The combination of the higher base pair stability at G-C regions and increased duplex DNA stability induced by intercalating antibiotic yields a total additive stability of the intercalator-G-C base pair complex that resists helicase action.

Animals↗
PubMed1992

A comparative study of 0.25% ropivacaine and 0.25% bupivacaine for brachial plexus block.

The present study compares the effectiveness of 0.25% ropivacaine and 0.25% bupivacaine in 44 patients receiving a subclavian perivascular brachial plexus block for upper extremity surgery. The patients were assigned to two equal groups in this randomized, double-blind study; one group received ropivacaine 0.25% (112.5 mg) and the other, bupivacaine 0.25% (112.5 mg), both without epinephrine. Onset times for analgesia and anesthesia in each of the C-5 through T-1 brachial plexus dermatomes did not differ significantly between the two groups. The mean onset time for analgesia ranged from 11.2 to 20.2 min, and the mean onset time for anesthesia ranged from 23.3 to 48.2 min. The onset of motor block differed only with respect to paresis in the hand, with bupivacaine demonstrating a shorter onset time than ropivacaine. The duration of sensory and motor block also was not significantly different between the two groups. The mean duration of analgesia ranged from 9.2 to 13.0 h, and the mean duration of anesthesia ranged from 5.0 to 10.2 h. Both groups required supplementation with peripheral nerve blocks or general anesthesia in a large number of cases, with 9 of the 22 patients in the bupivacaine group and 8 of the 22 patients in the ropivacaine group requiring supplementation to allow surgery to begin. In view of the frequent need for supplementation noted with both 0.25% ropivacaine and 0.25% bupivacaine, we do not recommend using the 0.25% concentrations of these local anesthetics to provide brachial plexus block.

Adult↗
PubMed1992

Helicase inhibition by anthracycline anticancer agents.

Helicases are essential to both DNA replication and transcription because they separate double-stranded DNA, preparing the single strands for replication or transcription. Because the anti-cancer anthracycline antibiotics stabilize double-stranded DNA primarily by their intercalative binding, we expected the intercalated antibiotics to interfere with helicase action. We examined anthracycline antibiotic effects on SV40 large T antigen helicase activity, using a duplex DNA helicase substrate of 32P-labeled 17-mer annealed to complementary M13mp19(+) circular single-stranded DNA. The T antigen helicase activity was potently inhibited by the anthracycline antibiotics. The T antigen helicase IC50 values for the anthracycline antibiotics were as follows: nogalamycin, 2 x 10(-7) M; daunorubicin, 4 x 10(-7) M; doxorubicin, 4 x 10(-7) M; idarubicin, 1.8 x 10(-6) M; 4'-epidoxorubicin, 2 x 10(-6) M; aclacinomycin, 4 x 10(-6) M; and menogaril, 6 x 10(-6) M. Partially purified helicases from HeLa cells and murine mammary carcinoma FM3A cells also were potently inhibited by doxorubicin, with IC50 values of 4 x 10(-7) M and 9 x 10(-7) M, respectively. Because the abundance, specificities, and types of helicases vary in the cell, this site of action for anthracycline antibiotics may help explain anthracycline potency, drug specificity for DNA or RNA inhibition, and some types of cellular resistance to these drugs.

Animals↗
PubMed1991

A dominant positive and negative selectable gene for use in mammalian cells.

We have constructed three different fusion genes containing the herpes simplex virus thymidine kinase (HSV tk) and the bacterial neomycin phosphotransferase (neo) genes. All three fusion genes utilize the HSV tk promoter but differ at the junction of their components. We have determined if the fusion genes are bifunctional by introducing them into mammalian cells and testing for function of the individual components. One of the fusion genes, TNFUS 69, produced a bicistronic message and a fusion protein that has TK and NEO protein functions. This and other fusion genes of a similar nature could serve as dominant positive and negative selectable markers in mammalian cells.

Amino Acid Sequence↗
PubMed1991

A comparison of ropivacaine 0.5% and bupivacaine 0.5% for brachial plexus block.

This study compared the effectiveness of 0.5% ropivacaine and 0.5% bupivacaine for brachial plexus block. Forty-eight patients received a subclavian perivascular brachial plexus block for upper-extremity surgery. One group (n = 24) received ropivacaine 0.5% (175 mg) and a second group (n = 24) received bupivacaine 0.5% (175 mg), both without epinephrine. Onset times for analgesia and anesthesia in each of the C5 through T1 brachial plexus dermatomes did not differ significantly between groups. Duration of analgesia and anesthesia was long (mean duration of analgesia, 13-14 h; mean duration of anesthesia, 9-11 h) and also did not differ significantly between groups. Motor block was profound, with shoulder paralysis as well as hand paresis developing in all of the patients in both groups. Two patients in each group required supplemental blocks before surgery. Ropivacaine 0.5% and bupivacaine 0.5% appeared equally effective in providing brachial plexus anesthesia.

Adult↗
PubMed1990

Evidence for DNA binding activity of numatrin (B23), a cell cycle-regulated nuclear matrix protein.

Stimulation of various cell types with growth factors is associated with a rapid induction in the synthesis of a nuclear matrix protein, termed 'numatrin' which was shown to be identical to the nucleolar protein B23. The abundance of numatrin was shown to be correlated with entry and progression through the S-phase. Thus, experiments were undertaken to examine whether numatrin also has DNA binding activity. Using whole nuclear extract, we showed that numatrin binds to both double-stranded (DS) DNA and to single-stranded (SS) DNA cellulose columns. Purified numatrin, which was extracted either under native conditions (in oligomeric form) or under urea conditions (in monomeric form), demonstrated significant binding to either [3H]DS-DNA or [3H]DS-DNA as shown by nitrocellulose filter binding assay. However, numatrin binding to DS-DNA was qualitatively and quantitatively different from its binding to SS-DNA. Thus, the binding of numatrin was several fold higher to DS-DNA as compared to SS-DNA. The binding to DS-DNA was reduced by 77% in the presence of 0.5 M NaCl, while the binding to SS-DNA was not affected under this condition. Furthermore, treatment of the native numatrin under conditions which caused monomerization of the protein resulted in a significant enhancement of numatrin binding to SS-DNA but did not affect the binding to DS-DNA. Following heparin-Sepharose chromatography purification (under native conditions), numatrin at picomole amounts showed significant binding to both DS-DNA and SS-DNA. Finally, numatrin was found to copurify with the complex of DNA polymerase alpha primase together with other proteins required for SV-40 in vitro replication activity. These results demonstrate that numatrin has DNA binding activity, and imply a possible role for numatrin/B23 in DNA-associated processes.

Blotting, Western↗
PubMed1990

Patterns of failure in anorectal melanoma. A guide to surgical therapy.

Anorectal melanoma is an aggressive tumor with a reported 5-year survival rate of 6%. Recommendations for local surgical therapy vary from local excision to abdominoperineal resection. Therapy, patterns of failure, and survival were retrospectively examined in 32 patients with anorectal melanoma. Twenty-six patients were treated surgically, 14 with abdominoperineal resection and 12 with local excision. Local recurrence occurred less frequently in patients undergoing abdominoperineal resection (4 [29%] of 14) compared with patients undergoing local excision (7 [58%] of 12) but developed concomitantly with distant or regional metastasis in all but 2 of the 11 patients whose operations failed locally. Inguinal nodal disease developed in 15 patients (47%). Pelvic nodal disease became apparent in only 2 patients (7%). There was no difference in overall survival between the two surgically treated groups (median survival, 19.5 months for patients treated with abdominoperineal resection vs 18.9 months for patients treated with local excision). Therefore, local excision is recommended when technically feasible since these patients eventually succumb to metastasis regardless of surgical therapy.

Anal Canal↗
PubMed1990

Brachial plexus block with a new local anaesthetic: 0.5 per cent ropivacaine.

A new local anaesthetic, ropivacaine hydrochloride, was used in a concentration of 0.5 per cent in 32 patients receiving a subclavian perivascular block for upper extremity surgery. One group (n = 15) received 0.5 per cent ropivacaine without epinephrine and a second group (n = 17) received 0.5 per cent ropivacaine with epinephrine in a concentration of 1:200,000. Anaesthesia was achieved in 87 per cent of the patients in both groups in all of the C5 through T1 brachial plexus dermatomes. Motor block was profound with 100 per cent of patients in both groups developing paresis at both the shoulder and hand and 100 per cent developing paralysis at the shoulder. There was a rapid initial onset of sensory block (a mean of less than four minutes for analgesia) with a prolonged duration (a mean of greater than 13 hr of analgesia). The addition of epinephrine did not significantly affect the quality or onset of sensory or motor block. The duration of sensory block was reduced by epinephrine at T1 for analgesia and at C7, C8, and T1 for anaesthesia. The duration of sensory block in the remaining brachial plexus dermatomes as well as the duration of motor block was not effected by epinephrine. There was no evidence of cardiovascular or central nervous system toxicity in either group with a mean dose of 2.5-2.6 mg.kg-1 ropivacaine.

Adult↗
PubMed1990

Plasma concentrations of ropivacaine given with or without epinephrine for brachial plexus block.

The purpose of this study was to determine the pharmacokinetic properties of the local anaesthetic ropivacaine used with or without epinephrine for brachial plexus block. Seventeen ASA physical status I or II adult patients undergoing elective orthopaedic surgery received a single injection of 33 ml ropivacaine for subclavian perivascular block and 5 ml to block the intercostobrachial nerve in the axilla. One group (n = 8) received 0.5 per cent ropivacaine without epinephrine (190 mg) and the other (n = 9) received 0.5 per cent ropivacaine with epinephrine 1:200,000 (190 mg). Plasma ropivacaine concentrations were measured from peripheral venous blood samples taken for 12 hr after drug administration. Ropivacaine base was determined in plasma using gas chromatography and a nitrogen-sensitive detector. The mean peak plasma concentration (Cmax) was 1.6 +/- 0.6 mg.L-1 and 1.3 +/- 0.4 mg.L-1 after administration of ropivacaine with and without epinephrine. The median time to peak plasma concentration (tmax) was 0.75 hr and 0.88 hr and the mean area under the plasma concentration curve AUC0-12h was 7.7 +/- 3.6 and 7.0 +/- 3.4 mg.l hr-1. The differences were not statistically significant. The terminal phase of the individual plasma concentration-time curves showed a varying and sometimes slow decline possibly indicating a sustained systemic uptake of ropivacaine from the brachial plexus. No central nervous system or cardiovascular symptoms attributed to systemic plasma concentrations of the drug were observed, with the dose (1.90-3.28 mg.kg-1) of ropivacaine used. It is concluded that the addition of epinephrine does not alter the pharmacokinetic properties of ropivacaine when used for subclavian perivascular brachial plexus block.

Adult↗