bluuebunny/crossref_metadata_2025 huggingface dataset:
The ATPase and protease domains of yeast mitochondrial Lon: Roles in proteolysis and respiration-dependent growth
Authors: van Dijl Jan Maarten , Kutejová Eva , Suda Kitaru , Perečko Dušan , Schatz Gottfried , Suzuki Carolyn K.
Published: July 2002

The ATP-dependent Lon protease of
Saccharomyces cerevisiae
mitochondria is required for selective proteolysis in the matrix, maintenance of mitochondrial DNA, and respiration-dependent growth. Lon may also possess a chaperone-like function that facilitates protein degradation and protein-complex assembly. To understand the influence of Lon’s ATPase and protease activities on these functions, we examined several Lon mutants for their ability to complement defects of Lon-deleted yeast cells. We also developed a rapid procedure for purifying yeast Lon to homogeneity to study the enzyme’s activities and oligomeric state. A point mutation in either the ATPase or the protease site strongly inhibited the corresponding activity of the pure protein but did not alter the protein’s oligomerization; when expressed at normal low levels neither of these mutant enzymes supported respiration-dependent growth of Lon-deleted cells. When the ATPase- or the protease-containing regions of Lon were expressed as separate truncated proteins, neither could support respiration-dependent growth of Lon-deleted cells; however, coexpression of these two separated regions sustained wild-type growth. These results suggest that yeast Lon contains two catalytic domains that can interact with one another even as separate proteins, and that both are essential for the different functions of Lon.
Suppression of adenovirus E1A-induced apoptosis by mutated p53 is overcome by coexpression with Id proteins
Authors: Nakajima Takuma , Yageta Mika , Shiotsu Kohhei , Morita Kenichi , Suzuki Mitsuhiro , Tomooka Yasuhiro , Oda Kinichiro
Published: July 2002

The rat 3Y1 derivative cell lines, EId10 and EId23, established by introducing the adenovirus E1A12S, Id-1H, and Id-2H cDNAs linked to the hormone-inducible promoter, express these proteins upon treatment with dexamethasone and elicit apoptosis, although these cell lines express mutated p53. The E1A mutants containing a deletion in either the N terminus or the conserved region 1 were unable to induce apoptosis in cooperation with Ids. Western blot analysis of the immunoprecipitates prepared from the dexamethasone-treated EId10 cell extract showed that Id-2H preferentially binds to E1A and E2A (E12/E47) helix–loop–helix transcription factorsin vivo, but scarcely to the retinoblastoma protein. After induction of E1A and Ids, EId10 and EId23 cells began to accumulate in S phase and undergo apoptosis before entering G2phase, suggesting that abnormal synthesis of DNA induced by coexpression of E1A, Id-1H, and Id-2H results in the induction of apoptosis. Apoptosis also is induced in mouse A40 (p53−/−) cells by E1A alone or E1A plus Ids after transient transfection of the expression vectors. The induction of apoptosis is stimulated by coexpression with wild-type p53; however, apoptosis induced by E1A alone was suppressed completely by coexpression with mutated p53, whereas apoptosis induced by E1A plus Ids was stimulated by the mutated p53 as done by wild-type p53. These results suggest that the suppressive function of mutated p53 is overcome by Ids.
DNase I-hypersensitive sites I and II of the human growth hormone locus control region are a major developmental activator of somatotrope gene expression
Authors: Bennani-Baïti Idriss M. , Asa Sylvia L. , Song Decheng , Iratni Rabah , Liebhaber Stephen A. , Cooke Nancy E.
Published: July 2002

High-level expression of the human growth hormone (hGH) gene is limited to somatotrope and lactosomatotrope cells of the anterior pituitary. We previously identified a locus control region (LCR) for the hGH gene composed of four tissue-specific DNase I-hypersensitive sites (HS) located between −14.6 kb and −32 kb 5′ to the hGH transcription start site that is responsible for establishing a physiologically regulated chromatin domain for hGH transgene expression in mouse pituitary. In the present study we demonstrated that the LCR mediates somatotrope and lactosomatotrope restriction on an otherwise weakly and diffusely expressed hGH transgene. The subregion of the LCR containing the two pituitary-specific HS, HSI and HSII (−14.6 to −16.2 kb relative to the hGH promoter and denoted HSI,II), was found to be sufficient for mediating somatotrope and lactosomatotrope restriction, for appropriately timed induction of hGH transgene expression between embryonic days 15.5 and 16.5, and for selective extinction of hGH expression in mature lactotropes. When studied by cell transfection, the HSI,II fragment selectively enhanced transcription in a presomatotrope-derived cell line, although at levels (2- to 3-fold) well below that seen
in vivo
. The LCR activity of the HSI,II element was therefore localized by scoring transgene expression in fetal founder pituitaries at embryonic day 18.5. The data from these studies indicated that a 404-bp segment of the HSI,II region encodes a critical subset of LCR functions, including the establishment of a productive chromatin environment, cell-specific restriction and enhancement of expression, and appropriately timed induction of the hGH transgene during embryonic development.
Mass spectrometric and capillary electrophoretic investigation of the enzymatic degradation of heparin-like glycosaminoglycans
Authors: Rhomberg Andrew J. , Ernst Steffen , Sasisekharan Ram , Biemann Klaus
Published: July 2002

Difficulties in determining composition and sequence of glycosaminoglycans, such as those related to heparin, have limited the investigation of these biologically important molecules. Here, we report methodology, based on matrix-assisted laser desorption ionization MS and capillary electrophoresis, to follow the time course of the enzymatic degradation of heparin-like glycosaminoglycans through the intermediate stages to the end products. MS allows the determination of the molecular weights of the sulfated carbohydrate intermediates and their approximate relative abundances at different time points of the experiment. Capillary electrophoresis subsequently is used to follow more accurately the abundance of the components and also to measure sulfated disaccharides for which MS is not well applicable. For those substrates that produce identical or isomeric intermediates, the reducing end of the carbohydrate chain was converted to the semicarbazone. This conversion increases the molecular weight of all products retaining the reducing terminus by the “mass tag” (in this case 56 Da) and thus distinguishes them from other products. A few picomoles of heparin-derived, sulfated hexa- to decasaccharides of known structure were subjected to heparinase I digestion and analyzed. The results indicate that the enzyme acts primarily exolytically and in a processive mode. The methodology described should be equally useful for other enzymes, including those modified by site-directed mutagenesis, and may lead to the development of an approach to the sequencing of complex glycosaminoglycans.
Reactivation of
XIST
in normal fibroblasts and a somatic cell hybrid: Abnormal localization of XIST RNA in hybrid cells
Authors: Hansen R. Scott , Canfield Theresa K. , Stanek Ann M. , Keitges Elizabeth A. , Gartler Stanley M.
Published: July 2002

The
XIST
gene, expressed only from the inactive X chromosome, is a critical component of X inactivation. Although apparently unnecessary for maintenance of inactivation,
XIST
expression is thought to be sufficient for inactivation of genes in cis even when
XIST
is located abnormally on another chromosome. This repression appears to involve the association of XIST RNA with the chromosome from which it is expressed. Reactivated genes on the inactive X chromosome, however, maintain expression in several somatic cell hybrid lines with stable expression of
XIST
. We describe here another example of an
XIST
-expressing human–hamster hybrid that lacks X-linked gene repression in which the human
XIST
gene present on an active X chromosome was reactivated by treatment with 5-aza-2′-deoxycytidine. These data raise the possibility that human XIST RNA does not function properly in human–rodent somatic cell hybrids. As part of our approach to address this question, we reactivated the
XIST
gene in normal male fibroblasts and then compared their patterns of XIST RNA localization by subcellular fractionation and
in situ
hybridization with those of hybrid cells. Although XIST RNA is nuclear in all cell types, we found that the
in situ
signals are much more diffuse in hybrids than in human cells. These data suggest that hybrids lack components needed for XIST localization and, presumably, XIST-mediated gene repression.
Lipophilic siderophores of
Mycobacterium tuberculosis
prevent cardiac reperfusion injury
Authors: Horwitz Lawrence D. , Sherman Nancy A. , Kong Yinong , Pike Adrian W. , Gobin Jovana , Fennessey Paul V. , Horwitz Marcus A.
Published: July 2002

Reperfusion injury, which occurs upon the reintroduction of blood
flow to an ischemic organ, is responsible for considerable damage in
heart attacks and strokes. However, no treatment for reperfusion injury
is currently available. A major cause of reperfusion injury is the
iron-mediated generation of hydroxyl radical (⋅OH). In this study
we have explored the capacity of novel iron chelators called
“exochelins” to prevent reperfusion injury. Exochelins,
siderophores of
Mycobacterium tuberculosis
, are unique
iron chelators because they are lipid soluble, and hence able to enter
cells rapidly. In the iron-free state, exochelins prevented ⋅OH
formation. Desferri-exochelins prevented oxidative injury to cultured
cardiac myocytes, and did so more rapidly and effectively than the
nonlipid soluble iron chelator deferoxamine. The capacity of various
desferri-exochelins to protect myocytes from oxidative injury varied
directly with their solubility in lipid. Infused into isolated rabbit
hearts during reperfusion after a period of ischemia,
desferri-exochelins dramatically improved systolic and diastolic left
ventricular function, preserved coronary flow, reduced release of the
cardiac enzyme lactic dehydrogenase, and reduced myocardial
concentrations of ⋅OH metabolites. Thus, highly diffusible
desferri-exochelins block injury caused by ⋅OH production and have
potential for the treatment of reperfusion injury.
Plant ribosome recycling factor homologue is a chloroplastic protein and is bactericidal in
Escherichia coli
carrying temperature-sensitive ribosome recycling factor
Authors: Rolland Norbert , Janosi Laszlo , Block Maryse A. , Shuda Masahiro , Teyssier Emeline , Miège Christine , Chéniclet Catherine , Carde Jean-Pierre , Kaji Akira , Joyard Jacques
Published: July 2002

We have isolated a protein, mature RRFHCP, from chloroplasts of spinach (
Spinacia oleracea
L.) that shows 46% sequence identity and 66% sequence homology with ribosome recycling factor (RRF) of
Escherichia coli
. RRF recycles ribosomes through disassembly of the posttermination complex. From the cDNA analysis and from the amino-terminal sequencing of the isolated protein, the mature RRFHCP was deduced to have a
M
r
of 21,838 with 193 aa. It lacks the 78-aa chloroplast targeting sequence encoded by the RRFHCP cDNA sequence. The RRFHCP synthesized
in vitro
was imported into isolated chloroplasts with simultaneous conversion to the mature RRFHCP. Transcription of the gene coding for RRFHCP was not dependent on light, yet it was limited mostly to photosynthetic tissues in which only one transcript size was detected. Mature RRFHCP exerted a bactericidal effect on
E. coli
carrying temperature-sensitive RRF at the permissive temperature whereas wild-type
E. coli
was not affected.
NIH shift in flavin-dependent monooxygenation: Mechanistic studies with 2-aminobenzoyl-CoA monooxygenase/reductase
Authors: Hartmann Steffen , Hultschig Claus , Eisenreich Wolfgang , Fuchs Georg , Bacher Adelbert , Ghisla Sandro
Published: July 2002

The flavoprotein 2-aminobenzoyl-CoA monooxygenase/reductase from the eubacterium
Azoarcus evansii
catalyzes the dearomatization of 2-aminobenzoyl-CoA. The reaction consists in an O
2
-dependent monooxygenation at the benzene position 5, which is followed immediately by an NADH-dependent hydrogenation of the intermediate at the same catalytic locus. The reaction was studied by
1
H,
2
H, and
13
C NMR spectroscopy of the products. The main product was characterized as 5-oxo-2-aminocyclohex-1-ene-1-carboxyl-CoA by two-dimensional NMR spectroscopy. Thus, [5-
2
H]2-aminobenzoyl-CoA was converted into [6-
2
H]5-oxo-2-aminocyclohex-1-ene-1-carboxyl-CoA, indicating a 5 → 6 shift of the [5-
2
H] label. Label from NAD
2
H was transferred to the 3 position of the cyclic eneamine, whereas label from solvent D
2
O was incorporated into the 4 and the 6 positions of 5-oxo-2-aminocyclohex-1-ene-1-carboxyl-CoA. The labeling pattern is compatible with the monooxygenation proceeding via what is formally an NIH shift, yielding 5-oxo-2-aminocyclohex-1,3-diene-1-carboxyl-CoA as a protein-bound intermediate. It is suggested that this shift in flavin-dependent monooxygenation may have general validity.
Adhesive properties of the isolated amino-terminal domain of platelet glycoprotein Ibα in a flow field
Authors: Marchese Patrizia , Saldívar Enrique , Ware Jerry , Ruggeri Zaverio M.
Published: July 2002

We have examined the interaction between the amino-terminal domain of platelet glycoprotein (GP) Ibα and immobilized von Willebrand Factor (vWF) under flow conditions in the absence of other components of the GP Ib–IX–V complex. Latex beads were coated with a recombinant fragment containing GP Ibα residues 1–302, either with normal sequence or with the single G233V substitution that causes enhanced affinity for plasma vWF in platelet-type pseudo-von-Willebrand disease. Beads coated with native fragment adhered to vWF in a manner comparable to platelets, showing surface translocation that reflected the transient nature of the bonds formed. Thus, the GP Ibα extracellular domain is necessary and sufficient for interacting with vWF under high shear stress. Beads coated with the mutated fragment became tethered to vWF in greater number and had lower velocity of translocation than beads coated with the normal counterpart, suggesting that the G233V mutation lowers the rate of bond dissociation. Our findings define an approach for studying the biomechanical properties of the GP Ibα–vWF bond and suggest that this interaction is tightly regulated to allow rapid binding at sites of vascular injury, while permitting the concurrent presence of receptor and ligand in the circulation.
Suppression of apoptosis signal-regulating kinase 1-induced cell death by 14-3-3 proteins
Authors: Zhang Lixin , Chen Jing , Fu Haian
Published: July 2002

Apoptosis signal-regulating kinase 1 (ASK1) is a pivotal component of a signaling pathway induced by many death stimuli, including tumor necrosis factor α, Fas, and the anticancer drugs cisplatin and paclitaxel. Here we report that ASK1 proapoptotic activity is antagonized by association with 14-3-3 proteins. We found that ASK1 specifically bound 14-3-3 proteins via a site involving Ser-967 of ASK1. Interestingly, overexpression of 14-3-3 in HeLa cells blocked ASK1-induced apoptosis whereas disruption of the ASK1/14-3-3 interaction dramatically accelerated ASK1-induced cell death. Targeting of ASK1 by a 14-3-3-mediated survival pathway may provide a novel mechanism for the suppression of apoptosis.
Water: Foldase activity in catalyzing polypeptide conformational rearrangements
Authors: Xu F. , Cross T. A.
Published: July 2002

Polypeptide conformer interconversion in a low dielectric environment is shown to be highly dependent on water concentration. Water increases this rate by 10
3
, apparently by catalyzing hydrogen bond exchange, and thereby presenting functional properties analogous to that of a foldase. This catalytic effect is demonstrated on the interconversion of a parallel gramicidin dimer into an antiparallel dimer. A Hill coefficient of 6.5 is observed, illustrating the highly cooperative nature of the process. Protein folding in nonpolar environs, such as the hydrophobic core of a protein or the hydrophobic domain of a lipid bilayer, may be contingent on and rate-limited by the scarcity of water.
The HNF-4/HNF-1α transactivation cascade regulates gene activity and chromatin structure of the human serine protease inhibitor gene cluster at 14q32.1
Authors: Rollini Pierre , Fournier R. E. K.
Published: July 2002

Hepatocyte-specific expression of the α1-antitrypsin (α1AT) gene requires the activities of two liver-enriched transactivators, hepatocyte nuclear factors 1α and 4 (HNF-1α and HNF-4). The α1AT gene maps to a region of human chromosome 14q32.1 that includes a related serine protease inhibitor (serpin) gene encoding corticosteroid-binding globulin (CBG), and the chromatin organization of this ≈130-kb region, as defined by DNase I-hypersensitive sites, has been described. Microcell transfer of human chromosome 14 from fibroblasts to rat hepatoma cells results in activation of α1AT and CBG transcription and chromatin reorganization of the entire locus. To assess the roles of HNF-1α and HNF-4 in gene activation and chromatin remodeling, we transferred human chromosome 14 from fibroblasts to rat hepatoma cell variants that are deficient in expression of HNF-1α and HNF-4. The variant cells failed to activate either α1AT or CBG transcription, and chromatin remodeling failed to occur. However, α1AT and CBG transcription could be rescued by transfecting the cells with expression plasmids encoding HNF-1α or HNF-4. In these transfectants, the chromatin structure of the entire α1AT/CBG locus was reorganized to an expressing cell-typical state. Thus, HNF-1α and HNF-4 control both chromatin structure and gene activity of two cell-specific genes within the serpin gene cluster at 14q32.1.
Mechanism-based inhibition of the melatonin rhythm enzyme: Pharmacologic exploitation of active site functional plasticity
Authors: Khalil Ehab M. , De Angelis Jacqueline , Ishii Makoto , Cole Philip A.
Published: July 2002

Serotonin
N
-acetyltransferase is the enzyme responsible for the diurnal rhythm of melatonin production in the pineal gland of animals and humans. Inhibitors of this enzyme active in cell culture have not been reported previously. The compound
N
-bromoacetyltryptamine was shown to be a potent inhibitor of this enzyme
in vitro
and in a pineal cell culture assay (IC
50
≈ 500 nM). The mechanism of inhibition is suggested to involve a serotonin
N
-acetyltransferase-catalyzed alkylation reaction between
N
-bromoacetyltryptamine and reduced CoA, resulting in the production of a tight-binding bisubstrate analog inhibitor. This alkyltransferase activity is apparently catalyzed at a functionally distinct site compared with the acetyltransferase activity active site on serotonin
N
-acetyltransferase. Such active site plasticity is suggested to result from a subtle conformational alteration in the protein. This plasticity allows for an unusual form of mechanism-based inhibition with multiple turnovers, resulting in “molecular fratricide.”
N
-bromoacetyltryptamine should serve as a useful tool for dissecting the role of melatonin in circadian rhythm as well as a potential lead compound for therapeutic use in mood and sleep disorders.
Novel Ras antagonist blocks human melanoma growth
Authors: Jansen B. , Schlagbauer-Wadl H. , Kahr H. , Heere-Ress E. , Mayer B. X. , Eichler H.-G. , Pehamberger H. , Gana-Weisz M. , Ben-David E. , Kloog Y. , Wolff K.
Published: July 2002

During past decades, knowledge of melanoma biology has increased considerably. Numerous therapeutic modalities based on this knowledge are currently under investigation. Advanced melanoma, nevertheless, remains a prime example of poor treatment response that may, in part, be the consequence of activated N-Ras oncoproteins. Besides oncogenic Ras, wild-type Ras gene products also play a key role in receptor tyrosine kinase growth factor signaling, known to be of importance in oncogenesis and tumor progression of a variety of human neoplasms, including malignant melanoma; therefore, it is reasonable to speculate that a pharmacological approach that curtails Ras activity may represent a sensible approach to inhibit melanoma growth. To test this concept, the antitumor activity of S-
trans
,
trans
-farnesylthiosalicylic acid (FTS), a recently discovered Ras antagonist that dislodges Ras from its membrane-anchoring sites, was evaluated. The antitumor activity of FTS was assessed both
in vitro
and
in vivo
in two independent SCID mouse xenotransplantation models of human melanoma expressing either wild-type Ras (cell line 518A2) or activated Ras (cell line 607B). We show that FTS (5–50 μM) reduces the amounts of activated N-Ras and wild-type Ras isoforms both in human melanoma cells and Rat-1 fibroblasts, interrupts the Ras-dependent extracellular signal-regulated kinase in melanoma cells, inhibits the growth of N-Ras-transformed fibroblasts and human melanoma cells
in vitro
and reverses their transformed phenotype. FTS also causes a profound and statistically significant inhibition of 518A2 (82%) and 607B (90%) human melanoma growth in SCID mice without evidence of drug-related toxicity. Our findings stress the notion that FTS may qualify as a novel and rational treatment approach for human melanoma and possibly other tumors that either carry activated
ras
genes or rely on Ras signal transduction more heavily than nonmalignant cells.
Gene therapy to promote thromboresistance: Local overexpression of tissue plasminogen activator to prevent arterial thrombosis in an
in vivo
rabbit model
Authors: Waugh J. M. , Kattash M. , Li J. , Yuksel E. , Kuo M. D. , Lussier M. , Weinfeld A. B. , Saxena R. , Rabinovsky E. D. , Thung S. , Woo S. L. C. , Shenaq S. M.
Published: July 2002

Tissue-type plasminogen activator (tPA) catalyzes the rate-limiting initial step in the fibrinolytic cascade. Systemic infusion of tPA has become the standard of care for acute myocardial infarction. However, even the relatively short-duration protocols currently employed have encountered significant hemorrhagic complications, as well as complications from rebound thrombosis. Gene therapy offers a method of local high-level tPA expression over a prolonged time period to avoid both systemic hemorrhage and local rebound thrombosis. To examine the impact of local tPA overexpression, an adenoviral vector expressing tPA was created. The construct was characterized functionally
in vitro
, and the function of the vector was confirmed
in vivo
by delivery to the rabbit common femoral artery. Systemic coagulation parameters were not perturbed at any of the doses examined. The impact of local overexpression of tPA on
in vivo
thrombus formation was examined subsequently in a stasis/injury model of arterial thrombosis. The construct effectively prevented arterial thrombosis in treated animals, whereas viral and nonviral controls typically developed occluding thrombi. This construct thus offers a viable technique for promoting a locally thromboresistant small-caliber artery.
Anaerobic chlorophyll isocyclic ring formation inRhodobacter capsulatusrequires a cobalamin cofactor
Authors: Gough Simon P. , Petersen Bent O. , Duus Jens Ø.
Published: July 2002

The isocyclic ring of bacteriochlorophyll (BChl) is formed by the conversion of Mg-protoporphyrin monomethyl ester (MPE) to protochlorophyllide (PChlide). Similarities revealed byblastsearches with the putative anaerobic MPE-cyclase BchE suggested to us that this protein also uses a cobalamin cofactor. We found that vitamin B12(B12)-requiring mutants of thebluEandbluBgenes ofRhodobacter capsulatus, grown without B12, accumulated Mg-porphyrins. Laser desorption/ionization time-of-flight (LDI-TOF) MS and NMR spectroscopy identified them as MPE and its 3-vinyl-8-ethyl (mvMPE) derivative. Anin vivoassay was devised for the cyclase converting MPE to PChlide. Cyclase activity in the B12-dependent mutants required B12but not protein synthesis. The following reaction mechanism is proposed for this MPE-cyclase reaction. Adenosylcobalamin forms the adenosyl radical, which leads to withdrawal of a hydrogen atom and formation of the benzylic-type 131-radical of MPE. Withdrawal of an electron gives the 131-cation of MPE. Hydroxyl ion attack on the cation gives 131-hydroxy-MPE. Withdrawal of three hydrogen atoms leads successively to 131-keto-MPE, its 132-radical, and cyclization to PChlide.
ATP-dependent structural change of the eukaryotic clamp-loader protein, replication factor C
Authors: Shiomi Yasushi , Usukura Jiro , Masamura Yusuke , Takeyasu Kunio , Nakayama Yoshikazu , Obuse Chikashi , Yoshikawa Hiroshi , Tsurimoto Toshiki
Published: July 2002

The eukaryotic DNA sliding clamp that keeps DNA polymerase engaged
at a replication fork, called proliferating cell nuclear antigen
(PCNA), is loaded onto the 3′ ends of primer DNA through its
interaction with a heteropentameric protein complex called replication
factor C (RFC). The ATPase activity of RFC is necessary for formation
of a functional PCNA clamp. In the present study, the sensitivity of
RFC to partial proteolysis is used to show that addition of ATP,
ATPγS, or ADP induces different structural changes in RFC. Direct
observation by electron microscopy reveals that RFC has a closed
two-finger structure called the U form in the absence of ATP. This is
converted into a more open C form on addition of ATP. In contrast, the
structural changes induced by ATPγS or ADP are limited. These results
suggest that RFC adapts on opened configuration intermediately after
ATP hydrolysis. We further observe that PCNA is held between the two
fingers of RFC and propose that the RFC structure change we observe
during ATP hydrolysis causes the attached PCNA to form its active
ring-like clamp on DNA.
The function of the human homolog of
Saccharomyces cerevisiae REV1
is required for mutagenesis induced by UV light
Authors: Gibbs Peter E. M. , Wang Xi-De , Li Ziqiang , McManus Terrence P. , McGregor W. Glenn , Lawrence Christopher W. , Maher Veronica M.
Published: July 2002

In
Saccharomyces cerevisiae
, most mutations induced by a wide range of mutagens arise during translesion replication employing the
REV1
gene product and DNA polymerase ζ. As part of an effort to investigate mammalian mutagenic mechanisms, we have identified cDNA clones of the human homologs of the yeast
REV
genes and examined their function in UV mutagenesis. Previously, we described the isolation of a human homolog of yeast
REV3
, the catalytic subunit of pol ζ, and here report the identification and sequence of a human homolog of yeast
REV1
. This gene was isolated by identifying an expressed sequence tag encoding a peptide with similarity to the C terminus of yeast Rev1p, followed by sequencing of the clone and retrieval of the remaining cDNA by 5′ rapid amplification of cDNA ends. The human gene encodes an expected protein of 1,251 residues, compared with 985 residues in the yeast protein. The proteins share two amino-terminal regions of ≈100 residues with 41% and 20% identity, a region of ≈320 residues with 31% identity, and a central motif in which 11 of 13 residues are identical. Human cells expressing high levels of an
hREV1
antisense RNA grew normally, and were not more sensitive to the cytotoxic effect of 254 nm UV radiation than cells lacking antisense RNA. However, the frequencies of 6-thioguanine resistance mutants induced by UV in the cells expressing antisense hREV1 RNA were significantly lower than in the control (
P
= 0.01), suggesting that the human gene has a function similar to that of the yeast homolog.
Minority Situation Attitudes and Developments after the return to power of “Post-Communists” in Poland*
Authors: Majewicz Alfred F.
Published: July 2002

One of the most frequently heard phrases in Communist Poland was the notoriousnie ma“there is not, is not available, is out of stock,” reflecting the permanent shortage—of virtually everything—that was so characteristic of what was labeled “real socialism.” The phrase disappeared shortly after the introduction of a market economy overnight filled the hitherto empty shelves with an abundance of goods which had been unheard of throughout the postwar decades. The phrase became almost forgotten—only to make a sudden and seemingly unexpected (although foreseen by some) comeback, when the “post-Communists” were returned to power by the September 1993 parliamentary elections. It may not (yet?) concern Warsaw, which devours an unfair share of what the country can produce, and it may not (yet?) concern other larger Polish cities, but across the countryside chronic shortage is often felt only too bitterly. In spite of this most alarming development, however, the strong opinion prevails that there can be no return to the old centrally steered socialist economy. The changes have gone too far, enabling the people to resist attempts at revising the impressive economic, social and political reforms in Poland.
Becoming scientific and becoming professional: towards moderating rationalism in the initial teacher education curriculum
Authors: Ovens Peter
Published: July 2002

The aim of this mainly theoretical article is to sketch a curriculum model, based upon Stenhouse (1975), for the education of primary school teachers with a focus on their teaching of science. Arising from practical concerns, the purpose is to outline a framework for creative compliance with the more retrogressive characteristics of the national requirements. A distinction between training and education leads to a conceptualization of education as a process of becoming. This is applied to aspects of science and to aspects of education separately, and then brought together to envisage a moderated approach to the teaching and the assessment of student teachers which could guide practice.
The National Curriculum balance sheet for Key Stage 2: a researcher's view
Authors: Galton Maurice
Published: July 2002

The National Curriculum had very specific objectives. From the beginning there have been claims that the failure to implement the programme in full was the fault of a ‘liberal’ educational establishment. This article looks at implementation from the perspective of a researcher who was engaged in classroom observation, mainly in primary schools, during the late 1980s and early 1990s. Among the consequences of the implementation strategies adopted have been negligible effects on teaching methods, falling standards and lack of increased curriculum continuity on transfer to Key Stage 3. The findings of this research suggest that recent calls for teachers to be mandated as to how to teach the curriculum will not overcome these problems.
The Dearing review of qualifications 16–19: attempting to reconcile the irreconcilable?
Authors: Cummings Andy
Published: July 2002

This article seeks to evaluate critically the proposals set out in Sir Ron Dearing's report published in March 1996 (Dearing, 1996). Despite being broadly welcomed across the political spectrum, most of the proposals have yet to be implemented. The General Election of 1997 delayed the process, with the start date for a partially reformed system now set at September 2000. While there have been some moves away from what Dearing recommended, his review remains the key policy text in this area, particularly as his consultations did much to educate the professional audience. The report has attracted thorough examination (Hodgson and Spours, 1997; Spours and Young, 1996; Young, 1998) and much will be drawn from such sources. However, the key argument will be made that within the Dearing Report, and within much of what has been written about it, there is a reluctance to engage with fundamental issues of curriculum philosophy and values. Hence what is proposed, while seeming reasonable and pragmatic, does in fact attempt to reconcile the irreconcilable. There is no evidence to suggest that the Labour government will address these issues. With the exception of the New Deal, the whole area of post‐compulsory education represents policy continuity rather than radical innovation (Hodgson and Spours, 1999). Paradoxically the greater freedom and voluntarism in this area does offer the possibility of innovation at a local level.
In vitro effects of fluticasone propionate on IL‐13 production by mitogen‐stimulated lymphocytes
Authors: Di Lorenzo Gabriele , Pacor Maria Luisa , Pellitteri Maria Esposito , Gangemi Sebastiano , Di Blasi Patrizia , Candore Giuseppina , Colombo Alfredo , Lio Domenico , Caruso Calogero
Published: July 2002

Background: Corticosteroid administration produces multiple immunomodulatory effects, including down‐regulation of cytokine production by CD4 T lymphocytes. Fluticasone propionate (FP) (Glaxo Smith&Kline, Greenford, UK), a highly lipophilic topical corticosteroid, has been shown to be safe and effective in the treatment of asthma and of both seasonal and perennial rhinitis.

Aims: To gain insight into the mechanisms of FP therapeutic effects, we evaluated interleukin (IL)‐13 (a type 2 cytokine that seemingly plays a pivotal role in allergic mechanisms) production by mitogen‐stimulated peripheral blood mononuclear cells (MNC)
in vitro, treated or not with FP.

Methods: MNC from 10 healthy subjects and 10 asthmatic atopic patients with Parietaria allergy were stimulated v/v with phytohaemagglutinin (PHA) (50 γ/ml) or with complete medium alone as a control. Culture supernatants, in vitro treated or not with 10-7 or 10-8 M FP, were collected after 48 or 72 h incubation. IL‐13 production was assessed by enzyme‐linked immunosorbent assay. In random selected samples, after 4 or 24 h of cell cultures, RNA was extracted and IL‐4 and IL‐5 reverse transcriptase‐polymerase chain reaction (RT‐PCR) products analyzed.

Results: At 48 h, there were no differences in IL‐13 concentration in PHA‐stimulated cultures between healthy subjects and asthmatic patients (93.6 ± 18.9 versus 111.0 ± 25.1 pg/ml). At 72 h, similar results were obtained (63.9 ± 3.0 versus 73.3 ± 2.5 pg/ml, respectively). At this time, however, IL‐13 concentrations were significantly decreased versus 48 h both in asthmatics
(p < 0.001) and in controls (p < 0.001). Treatment with 10-7 M FP significantly reduced IL‐13 production in healthy subjects and asthmatic patients both at 48 h
(93.6 ± 18.9 versus 50.50 ± 10.6 pg/ml, p < 0.001, and 111.0 ± 25.1 versus 59.3 ± 13.6 pg/ml, p < 0.001, respectively) and at 72 h (63.9 ± 9.6 versus 35.5 ± 4.4 pg/ml, p < 0.001, and 73.3 ± 8.0 versus 40.7 ± 4.5 pg/ml, p < 0.001, respectively). Similar results were obtained with 10-8 M FP at 48 and 72 h. Accordingly, evaluation of RT‐PCR products from selected cell samples showed a FP dosage‐dependent inhibition of IL‐4 and IL‐5 mRNA production both for healthy subjects and asthmatic patients.

Conclusions: FP in vitro impairs IL‐13 production by PHA‐stimulated MNC from asthmatic and control subjects. This strengthens previous suggestions that IL‐13 inhibition by steroids may, at least in part, account for their therapeutic effects.
The release of eosinophil chemotactic activity and eosinophil chemokinesis inhibitory activity by mononuclear cells from atopic asthmatic and non‐atopic subjects
Authors: Grzegorczyk J. , Majkowska-Wojciechowska B. , Kowalski M. L.
Published: July 2002

The goal of our study was to assess the chemotactic activity for eosinophils (ECA) and neutrophils (NCA) and histamine releasing activity (HRA) in crude supernatants of mononuclear cells in monosensitized atopic asthmatics and healthy controls. Chemotactic activity for ECA and neutrophils was measured in supernatants of cultured mononuclear cells with modified Boyden’s chamber and HRA was assessed on healthy donor basophils. With respect to ECA generation two distinct subgroups of subjects were distinguished: releasers [ECA (+)] and non‐releasers [ECA (–)]. In atopic and non‐atopic ECA (+) the mean ECA index was 3.78 ± 0.49 and 2.47 ± 0.27 respectively (P > 0.05). Supernatants from the remaining subjects (seven of 22 atopic and five of 11 non‐atopic) did not express ECA, but revealed significant inhibitory activity for chemokinesis of eosinophils (mean chemotactic index 0.25 ± 0.16 and 0.48 ± 0.22 for atopic and non‐atopic non‐releasers respectively). Stimulation with antigen of MNC from atopic and with PHA from non‐atopic ECA (–) restored cells ability to release ECA. Sephadex gel chromatography revealed that supernatants of MNC contained chemotactic and chemokinesis inhibitory activity in different fractions. The spontaneous productions of NCA and HRA by mononuclear cells was sim ilar in ECA releasers and non‐releasers, although the HRA was higher following stimulation with PHA in the non‐atopic ECA (+) subgroup. Our study demonstrated, for the first time, that MNC are capable of generating not only chemotactic activity but also chemokinesis inhibitory activity for eosinophils.
Suppressive activity of a macrolide antibiotic, roxithromycin on co‐stimulatory molecule expression on mouse splenocytes in vivo
Authors: Kawazu K. , Kurokawa M. , Asano K. , Mita A. , Adachi M.
Published: July 2002

The influence of roxithromycin (RXM) on the expression of co‐stimulatory molecules, CD40, CD80 and CD86, was examined in vivo. When BALB/c mice were immunized intraperitoneally with two doses of dinitrophenylated ovalbumin (DNP‐OVA) at 1 week intervals, intraperitoneal administration of RXM at 250 μg/kg once a day for 14 days strongly suppressed IgE contents in sera obtained from mice 22 days after the first immunization. In addition, RXM treatment of mice suppressed endogenous IL–4 contents in aqueous spleen extracts, which were enhanced by DNP‐OVA immunization. We next examined the influence of RXM on co‐stimulatory molecule expression on splenic lymphocytes. RXM treatment of the immunized mice caused suppression of CD40 expression, but this treatment did not affect CD80 and CD86 expression.
Improvement of nutrient absorption may enhance systemic oxidative stress in cystic fibrosis patients
Authors: Shmarina Galina V. , Pukhalsky Alexander L. , Kokarovtseva Svetlana N. , Pukhalskaya Daria A. , Kalashnikova Elena A. , Kapranov Nikolaï I. , Kashirskaja Natalia J.
Published: July 2002

Background: The life expectancy of patients with cystic fibrosis (CF) is largely dependent on the pulmonary disease severity and progress. Malnutrition may be an important complicating factor in active and chronic lung disease.

Aims: The focus of this study was to investigate several inflammatory markers in pancreatic‐insufficient CF patients with different enzyme treatment regimens.

Methods: CF patients with pancreatic insufficiency were examined at a time of symptomatic exacerbation of their lung disease. Group A (n = 11) regularly received microspheric enzymes. Group B (n = 8) were treated with enzymes during the hospitalization period only and demonstrated the presence of malnutrition. Inflammatory markers in the sputa (neutrophil elastase activity, interleukin‐8 and tumour necrosis factor‐α levels) and in the peripheral blood (plasma malondialdehyde (MDA), lymphocyte response to PHA, and the cell sensitivity to steroid suppression) have been investigated.

Results: During acute lung exacerbation, group B demonstrated reduced levels of lymphocyte proliferation. This parameter was normalized after combined antibiotic and pancreatic enzyme therapy. Simultaneously, plasma MDA in group B markedly increased following treatment. For this group, a significant positive linear association between values of plasma MDA and lymphocyte proliferation has been observed. For group A, neither the same correlation nor changes in MDA levels and lymphocyte proliferation have been found.

Conclusions: Our data indicate that acute lung exacerbation in malnourished CF patients may be associated with alteration in T‐lymphocyte activity. Adequate therapy normalizes lymphocyte function but results in systemic oxidative stress.
Increased urinary nitrite, a marker of nitric oxide, in active inflammatory bowel disease
Authors: Goggins Michael G. , Shah Syed A. , Goh Jason , Cherukuri Anil , Weir Donal G. , Kelleher Dermot , Mahmud Nasir
Published: July 2002

Background: Nitric oxide (NO) production is increased in inflammatory bowel disease (IBD), and measurement of NO metabolites may be useful for monitoring disease activity.

Aims and objectives: To characterise urinary nitrite levels, a stable metabolite of NO, in IBD and to evaluate its potential as a marker of disease activity.

Methods: Twelve‐hour urinary nitrites were measured by the microplate assay method in 46 patients with IBD (active; n = 32). Urinary samples from 16 healthy individuals served as controls.

Results: Increased levels of urinary nitrites were found in patients with active IBD compared with those with inactive IBD. Twenty‐eight out of 32 patients (87.5%) with active IBD had detectable levels of nitrite in their urine as compared with 2/14 (14.3%) patients with inactive IBD. None of the 16 healthy controls had detectable urinary nitrite. Twelve‐hour urinary nitrite in active compared with inactive IBD: 5 Ī 0.7 versus 0.1 Ī 0.04 μ mol
(P < 0.05). There was good correlation between urinary nitrite and some markers of disease activity in IBD such as C‐reactive protein and microalbuminuria but not with erythrocyte sedimentation rate.

Conclusions: Increased levels of nitrite were detected in urine of patients with active IBD, consistent with increased NO synthesis. This simple assay may be exploited as a potential marker of disease
activity in IBD.
Release of tumor necrosis factor‐α and prostanoids in whole blood cultures after in vivo exposure to low‐dose aspirin
Authors: Beckmann Ilse , Ben-Efraim Shlomo , Vervoort Monica , Wallenburg Henk C. S.
Published: July 2002

Background: The preventive effect of low‐dose aspirin in cardiovascular disease is generally attributed to its antiplatelet action caused by differential inhibition of platelet cyclooxygenase‐1. However, there is evidence that aspirin also affects release of inflammatory cytokines, including tumor necrosis factor‐α (TNF‐α). It is not known whether this is caused by direct action on the cytokine pathway or indirectly through cyclooxygenase inhibition and altered prostanoid synthesis, or both.

Methods: We assessed the capacity of lipopolysaccharide‐activated leukocytes in whole blood cultures of eight healthy subjects following a single oral dose of 80 mg aspirin to release TNF‐α, prostanoid E2
(PGE2) and prostanoid I2 (PGI2), and thromboxane A2 (TXA2). TNF‐α and prostanoids were determined by enzyme‐linked immunoassays.

Results: In seven subjects, TNF‐α release in blood cultures decreased 24 h after intake of aspirin. The effect of aspirin on prostanoid release was assessed in three individuals: PGE2 increased in all subjects,
PGI2 increased in two and remained unchanged in one, and TXA2 was reduced in two and unchanged in one individua2l. The presence of DFU, a specific inhibitor of cyclooxygenase 2, did not affect the reduction of TNF‐α release by aspirin, but abolished prostanoid production in all three individuals.

Conclusion: The capacity of activated leukocytes to release TNF‐α is reduced by ingestion of low‐dose aspirin, independent of changes in prostanoid biosynthesis.
17β‐Estradiol promotes the synthesis and the secretion of annexin I in the CCRF‐CEM human cell line
Authors: Castro-Caldas Margarida , Duarte Carlos B. , Carvalho Arsélio P. , Lopes M. Celeste
Published: July 2002

Aims: Annexin I (ANXA1), a 37 kDa member of the annexin family of Ca2+‐binding and phospholipidbinding proteins, is particularly abundant in various populations of peripheral blood leukocytes. Since this protein modulates the anti‐inflammatory actions of the steroid hormones, the purpose of this study was to investigate the effects of the female sex steroid hormone, 17β‐estradiol (E2β), on the synthesis and secretion of ANXA1 in the human CCRF‐CEM acute lymphoblastic leukemia cell line.

Methods: Complementary reverse transcription‐polymerase chain reaction and Western blot assays were performed to study the effect of E2β on the expression of mRNA and protein ANXA1, respectively.

Results and discussion: Treatment of CCRF‐CEM cells with E2β, for 30 min, stimulated the synthesis of ANXA1 mRNA molecules, and increased the cellular level of ANXA1 protein. Moreover, when the cells were incubated with E2β, under the same experimental conditions, a significant increase in the amount of ANXA1 secreted from the cells was also detected. ICI 182,780, a selective inhibitor of the intracellular estrogen receptor, had no effect on the E2β‐stimulated expression and externalisation of ANXA1. Taken together, these results indicate that E2β induces de novo synthesis of ANXA1 and stimulates its secretion in the CCRF‐CEM cell line, apparently through a mechanism independent of the intracellular estrogen receptor.
Aggressive Fibromatosis: Evidence for a Stable Phase
Authors: Mitchell Gillian , Thomas J. Meirion , Harmer Clive L.
Published: July 2002

Purpose. Aggressive fibromatosis (AF) is an uncommon locally infiltrating benign disease of soft tissue for which treatment comprises complete surgical resection. Radiotherapy can be given postoperatively if the margin is incompletely resected. If the tumour is inoperable radiotherapy provides an alternative treatment. Hormone therapy and cytotoxic chemotherapy have also been used for unresectable or recurrent disease. All treatment modalities carry an associated morbidity. We believe that the natural history of aggressive fibromatosis may include a period of stable disease without progression, during which time, treatment is not always necessary.

Patients and methods. We present a retrospective review of 42 patients referred to the Royal Marsden Hospital between 1988 and 1995 with aggressive fibromatosis. Evidence of periods of stable disease and the relationship to delivered treatment was obtained from the case notes, including the natural history prior to referral to our institution. Stable disease was defined as a period of no objective progression for 6 months or longer.

Results. Seventeen patients could be assessed for stable disease and all (100%) experienced at least one episode of stable disease, eight of whom whilst receiving hormonal or cytotoxic therapy. Of the 23 patients who could not be assessed for stable disease, as they underwent surgery at presentation or recurrence of disease, only 2 had persisting disease at last follow‐up. Both of these patients had had positive surgical resection margins.

Discussion. This study demonstrates the variable natural history of AF, which can include a substantial period of stable disease in a significant number of patients. A less aggressive approach to the management of AF may therefore be appropriate, particularly if a subgroup of patients who are likely to experience a period of stable disease can be identified.
Radiotherapy for Lung Metastases in a Patient With Ewing Sarcoma
Authors: Kegye A. , Naszály A.
Published: July 2002

Purpose. To assess the benefit of therapy for multiple lung metastases in a young female patient previously treated with operation, local radiotherapy and chemotherapy.

Patient: Lung metastases occurred in a young female patient 13 months after finishing combined treatment of a Ewing sarcoma of the left eleventh rib. Primary treatment had included surgical removal, 51 Gy local radiotherapy and chemotherapy.

Method. 20 Gy total dose was applied to the lungs of both sides in two courses with an additional 15 Gy to the mediastinum.

Results and Discussion. Complete radiological regression was achieved at the end of therapy which was maintained during the follow‐up period of 16 months.