bluuebunny/crossref_metadata_2025 huggingface dataset:
Polyadenylation of stable RNA precursors
in vivo
Authors: Li Zhongwei , Pandit Shilpa , Deutscher Murray P.
Published: July 2002

Polyadenylation at the 3′ terminus has long been considered a specific feature of mRNA and a few other unstable RNA species. Here we show that stable RNAs in
Escherichia coli
can be polyadenylated as well. RNA molecules with poly(A) tails are the major products that accumulate for essentially all stable RNA precursors when RNA maturation is slowed because of the absence of processing exoribonucleases; poly(A) tails vary from one to seven residues in length. The polyadenylation process depends on the presence of poly(A) polymerase I. A stochastic competition between the exoribonucleases and poly(A) polymerase is proposed to explain the accumulation of polyadenylated RNAs. These data indicate that polyadenylation is not unique to mRNA, and its widespread occurrence suggests that it serves a more general function in RNA metabolism.
Interaction affinity between cytokine receptor components on the cell surface
Authors: Whitty Adrian , Raskin Natalya , Olson Dian L. , Borysenko Christopher W. , Ambrose Christine M. , Benjamin Christopher D. , Burkly Linda C.
Published: July 2002

The anti-common gamma chain (γc) mAb CP.B8 is shown to inhibit interleukin 4 (IL-4)-dependent proliferation of phytohemagglutinin (PHA) activated T cells noncompetitively with respect to cytokine by blocking the IL-4-induced heterodimerization of IL-4Rα and γcreceptor chains. Affinities for the binding of IL-4 to Cos-7 cells transfected with huIL-4Rα, and to PHA blasts expressing both IL-4Rα and γc, were used to estimate the affinity of the key interaction between γcand the binary IL-4Rα⋅IL-4 complex on the cell surface. This affinity was defined in terms of the dimensionless ratio [IL-4Rα⋅IL-4⋅γc]/[IL-4Rα⋅IL-4], which we designateKR. The results show that on PHA blasts this interaction is relatively weak;KR≈ 9, implying that ≈10% of the limiting IL-4Rα chain remains free of γceven at saturating concentrations of IL-4. This quantitative treatment establishesKRas a key measure of the coupling between ligand binding and receptor activation, providing a basis for functional distinctions between different receptors that are activated by ligand-induced receptor dimerization.
Oligomerization of activated
d
- and
l
-guanosine mononucleotides on templates containing
d
- and
l
-deoxycytidylate residues
Authors: Kozlov Igor A. , Pitsch Stefan , Orgel Leslie E.
Published: July 2002

The oligomerization of activated
d
- and
l
- and racemic guanosine-5′-phosphoro-2-methylimidazole on short templates containing
d
- and
l
-deoxycytidylate has been studied. Results obtained with
d
-oligo(dC)s as templates are similar to those previously reported for experiments with a poly(C) template. When one
l
-dC or two consecutive
l
-dCs are introduced into a
d
-template, regiospecific synthesis of 3′-5′ oligo(G)s proceeds to the end of the template, but three consecutive
l
-dCs block synthesis. Alternating
d
-,
l
-oligomers do not facilitate oligomerization of the
d
-,
l
-, and racemic 2-guanosine-5′-phosphoro-2-methylimidazole. We suggest that once a “predominately
d
-metabolism” existed, occasional
l
-residues in a template would not have led to the termination of self-replication.
Specific interaction of mutant p53 with regions of matrix attachment region DNA elements (MARs) with a high potential for base-unpairing
Authors: Will Katrin , Warnecke Gabriele , Wiesmüller Lisa , Deppert Wolfgang
Published: July 2002

Mutant, but not wild-type p53 binds with high affinity to a variety of MAR-DNA elements (MARs), suggesting that MAR-binding of mutant p53 relates to the dominant-oncogenic activities proposed for mutant p53. MARs recognized by mutant p53 share AT richness and contain variations of an AATATATTT “DNA-unwinding motif,” which enhances the structural dynamics of chromatin and promotes regional DNA base-unpairing. Mutant p53 specifically interacted with MAR-derived oligonucleotides carrying such unwinding motifs, catalyzing DNA strand separation when this motif was located within a structurally labile sequence environment. Addition of GC-clamps to the respective MAR-oligonucleotides or introducing mutations into the unwinding motif strongly reduced DNA strand separation, but supported the formation of tight complexes between mutant p53 and such oligonucleotides. We conclude that the specific interaction of mutant p53 with regions of MAR-DNA with a high potential for base-unpairing provides the basis for the high-affinity binding of mutant p53 to MAR-DNA.
Gap junctional coupling in lenses lacking α
3
connexin
Authors: Gong Xiaohua , Baldo George J. , Kumar Nalin M. , Gilula Norton B. , Mathias Richard T.
Published: July 2002

Fiber cells of the lens are interconnected by an extensive network of gap junctions containing α
3
(Cx46) and α
8
(Cx50) connexins. A specific role for these connexins in lens homeostasis is not known. To determine the contribution of these connexins to lens function, we used impedance techniques to study cell-to-cell coupling in lenses from homozygous α
3
knockout (−/−), heterozygous (+/−), and wild-type (+/+) mice. Western blots and immunofluorescence data indicated that α
8
remained at similar levels in the three classes of lenses, whereas α
3
was approximately 50% of the normal level in the +/− lenses, and it was absent from the −/− lenses. Moreover, the data from +/+ lenses suggest that a cleavage of connexins occurs abruptly between the peripheral shell of differentiating fibers (DF) and the inner core of mature fibers (MF). The appearance of the cleaved connexins was correlated to a change in the coupling conductance. In −/− lenses the coupling conductance of MF was zero, and these fibers were depolarized by about 30 mV from normal (≈−65 mV). The DF remained coupled, but the conductance was reduced to 30–35% of normal. However, the gap junctions in the DF of α
3
−/− lenses remained sensitive to pH. We conclude that α
3
connexin is necessary for the coupling of central fibers to peripheral cells, and that this coupling is essential for fiber cell homeostasis because uncoupled MF depolarize and subsequently become opaque.
Tyrosylprotein sulfotransferase: Purification and molecular cloning of an enzyme that catalyzes tyrosine
O
-sulfation, a common posttranslational modification of eukaryotic proteins
Authors: Ouyang Ying-bin , Lane William S. , Moore Kevin L.
Published: July 2002

Tyrosine
O
-sulfation is a common posttranslational modification of proteins in all multicellular organisms. This reaction is mediated by a Golgi enzyme activity called tyrosylprotein sulfotransferase (TPST) that catalyzes the transfer of sulfate from 3′-phosphoadenosine 5′-phosphosulfate to tyrosine residues within acidic motifs of polypeptides. Tyrosine
O
-sulfation has been shown to be important in protein–protein interactions in several systems. For example, sulfation of tyrosine residues in the leukocyte adhesion molecule P-selectin glycoprotein ligand 1 (PSGL-1) is required for binding to P-selectin on activated endothelium. In this report we describe the purification of TPST from rat liver microsomes based on its affinity for the N-terminal 15 amino acids of PSGL-1. We have isolated human and mouse TPST cDNAs that predict type II transmembrane proteins of 370 amino acid residues with almost identical primary structure. The human cDNA encodes a fully functional N-glycosylated enzyme with an apparent molecular mass of ≈54 kDa when expressed in mammalian cells. This enzyme defines a new class of Golgi sulfotransferases that may catalyze tyrosine
O
-sulfation of PSGL-1 and other protein substrates involved in diverse physiologic functions including inflammation and hemostasis.
IPF
2α
-I: An index of lipid peroxidation in humans
Authors: Praticò Domenico , Barry Orla P. , Lawson John A. , Adiyaman Mustafa , Hwang Seong-Woo , Khanapure Subhash P. , Iuliano Luigi , Rokach Joshua , FitzGerald Garret A.
Published: July 2002

Isoprostanes are prostaglandin isomers produced from arachidonic acid by a free radical-catalyzed mechanism. Urinary excretion of 8-
iso
-prostaglandin F
2α
, an isomer of the PGG/H synthase (cyclooxygenase or COX) enzyme product, prostaglandin F
2α
(PGF
2α
), has exhibited promise as an index of oxidant stress
in vivo.
We have developed a quantitative method to measure isoprostane F
2α
-I, (IPF
2α
-I) a class I isomer (8-
iso
-PGF
2α
is class IV), using gas chromatography/mass spectrometry. IPF
2α
-I is severalfold as abundant in human urine as 8-
iso-
PGF
2α
, with mean values of 737 ± 20.6 pg/mg creatinine. Both isoprostanes are formed in a free radical-dependent manner in low density lipoprotein oxidized by copper
in vitro
. However, IPF
2α
-I, unlike 8-
iso
-PGF
2α
, is not formed in a COX-dependent manner by platelets activated by thrombin or collagen
in vitro
. Similarly, COX inhibition
in vivo
has no effect on IPF
2α
-I. Neither serum IPF
2α
-I, an index of cellular capacity to generate the isoprostane, nor urinary excretion of IPF
2α
-I, an index of actual generation
in vivo
, is depressed by aspirin or indomethacin. In contrast, both serum thromboxane B
2
and urinary excretion of its 11-dehydro metabolite are depressed by the COX inhibitors. Although serum 8-
iso-
PGF
2α
formation is substantially depressed by COX inhibitors, urinary excretion of the compound is unaffected. Urinary IPF
2α
-I is elevated in cigarette smokers compared with controls (1525 ± 180 versus 740 ± 40 pg/mg creatinine;
P
< 0.01) and is highly correlated with urinary 8-
iso
-PGF
2α
(
r
= 0.9;
P
< 0.001). Urinary IPF
2α
-I is a novel index of lipid peroxidation
in vivo
, which can be measured with precision and sensitivity. It is an abundant F
2
-isoprostane formed in a free radical- but not COX-dependent manner. Although 8-
iso
-PGF
2α
may be formed as a minor product of COX, this pathway contributes trivially, if at all, to levels in urine. Urinary excretion of both isoprostanes is elevated in cigarette smokers.
Human
PEX1
cloned by functional complementation on a CHO cell mutant is responsible for peroxisome-deficient Zellweger syndrome of complementation group I
Authors: Tamura Shigehiko , Okumoto Kanji , Toyama Ryusuke , Shimozawa Nobuyuki , Tsukamoto Toshiro , Suzuki Yasuyuki , Osumi Takashi , Kondo Naomi , Fujiki Yukio
Published: July 2002

The peroxisome biogenesis disorders (PBDs), including Zellweger syndrome (ZS) and neonatal adrenoleukodystrophy (NALD), are autosomal recessive diseases caused by defects in peroxisome assembly, for which at least 10 complementation groups have been reported. We have isolated a human
PEX1
cDNA (
HsPEX1
) by functional complementation of peroxisome deficiency of a mutant Chinese hamster ovary (CHO) cell line, ZP107, transformed with peroxisome targeting signal type 1-tagged “enhanced” green fluorescent protein. This cDNA encodes a hydrophilic protein (Pex1p) comprising 1,283 amino acids, with high homology to the AAA-type ATPase family. A stable transformant of ZP107 with
HsPEX1
was morphologically and biochemically restored for peroxisome biogenesis.
HsPEX1
expression restored peroxisomal protein import in fibroblasts from three patients with ZS and NALD of complementation group I (CG-I), which is the highest-incidence PBD. A CG-I ZS patient (PBDE-04) possessed compound heterozygous, inactivating mutations: a missense point mutation resulting in Leu-664 → Pro and a deletion of the sequence from Gly-634 to His-690 presumably caused by missplicing (splice site mutation). Both PBDE-04
PEX1
cDNAs were defective in peroxisome-restoring activity when expressed in the patient fibroblasts as well as in ZP107 cells. These results demonstrate that
PEX1
is the causative gene for CG-I peroxisomal disorders.
Selection of functional T cell receptor mutants from a yeast surface-display library
Authors: Kieke Michele C. , Shusta Eric V. , Boder Eric T. , Teyton Luc , Wittrup K. Dane , Kranz David M.
Published: July 2002

The heterodimeric αβ T cell receptor (TCR) for antigen is the key determinant of T cell specificity. The structure of the TCR is very similar to that of antibodies, but the engineering of TCRs by directed evolution with combinatorial display libraries has not been accomplished to date. Here, we report that yeast surface display of a TCR was achieved only after the mutation of specific variable region residues. These residues are located in two regions of the TCR, at the interface of the α- and β-chains and in the β-chain framework region that is thought to be in proximity to the CD3 signal-transduction complex. The mutations are encoded naturally in many antibody variable regions, indicating specific functional differences that have not been appreciated between TCRs and antibodies. The identification of these residues provides an explanation for the inherent difficulties in the display of wild-type TCRs compared with antibodies. Yeast-displayed mutant TCRs bind specifically to the peptide/MHC antigen, enabling engineering of soluble T cell receptors as specific T cell antagonists. This strategy of random mutagenesis followed by selection for surface expression may be of general use in the directed evolution of other eukaryotic proteins that are refractory to display.
Poly-ADP ribose polymerase activates nuclear proteasome to degrade oxidatively damaged histones
Authors: Ullrich Oliver , Reinheckel Thomas , Sitte Nicolle , Hass Ralf , Grune Tilman , Davies Kelvin J. A.
Published: July 2002

The 20S proteasome has been shown to be largely responsible for the degradation of oxidatively modified proteins in the cytoplasm. Nuclear proteins are also subject to oxidation, and the nucleus of mammalian cells contains proteasome. In human beings, tumor cells frequently are subjected to oxidation as a consequence of antitumor chemotherapy, and K562 human myelogenous leukemia cells have a higher nuclear proteasome activity than do nonmalignant cells. Adaptation to oxidative stress appears to be one element in the development of long-term resistance to many chemotherapeutic drugs and the mechanisms of inducible tumor resistance to oxidation are of obvious importance. After hydrogen peroxide treatment of K562 cells, degradation of the model proteasome peptide substrate suc-LLVY-MCA and degradation of oxidized histones in nuclei increases significantly within minutes. Both increased proteolytic susceptibility of the histone substrates (caused by modification by oxidation) and activation of the proteasome enzyme complex occur independently during oxidative stress. This rapid up-regulation of 20S proteasome activity is accompanied by, and depends on, poly-ADP ribosylation of the proteasome, as shown by inhibitor experiments,
14
C-ADP ribose incorporation assays, immunoblotting,
in vitro
reconstitution experiments, and immunoprecipitation of (activated) proteasome with anti-poly-ADP ribose polymerase antibodies. The poly-ADP ribosylation-mediated activated nuclear 20S proteasome is able to remove oxidatively damaged histones more efficiently and therefore is proposed as an oxidant-stimulatable defense or repair system of the nucleus in K562 leukemia cells.
Duplex DNA catalyzes the chemical rearrangement of a malondialdehyde deoxyguanosine adduct
Authors: Mao Hui , Schnetz-Boutaud Nathalie C. , Weisenseel Jason P. , Marnett Lawrence J. , Stone Michael P.
Published: July 2002

The primary DNA lesion induced by malondialdehyde, a byproduct of lipid peroxidation and prostaglandin synthesis, is 3-(2′-deoxy-β-
d
-erythro-pentofuranosyl)-pyrimido[1,2-a]purin-10(3H)-one (M
1
G). When placed opposite cytosine (underlined) at neutral pH in either the d(GGTMTCCG)⋅d(CGGA
C
ACC) or d(ATCGCMCGGCATG)⋅ d(CATGCCG
C
GCGAT) duplexes, M
1
G spontaneously and quantitatively converts to the ring-opened derivative
N
2
-(3-oxo-1-propenyl)-dG. Ring-opening is reversible on thermal denaturation. Ring-opening does not occur at neutral pH in single-stranded oligodeoxynucleotides or when T is placed opposite to M
1
G in a duplex. The presence of a complementary cytosine is not required to stabilize
N
2
-(3-oxo-1-propenyl)-dG in duplex DNA at neutral pH. When
N
2
-(3-oxo-1-propenyl)-dG is placed opposite to thymine in a duplex, it does not revert to M
1
G. A mechanism for the conversion of M
1
G to
N
2
-(3-oxo-1-propenyl)-dG is proposed in which the exocyclic amino group of the complementary cytosine attacks the C8 position of the M
1
G exocyclic ring and facilitates ring opening via formation of a transient Schiff base. Addition of water to the Schiff base regenerates the catalytic cytosine and generates
N
2
-(3-oxo-1-propenyl)-dG. These results document the ability of duplex DNA to catalyze the transformation of one adduct into another, which may have important consequences for mutagenesis and repair.
Regulation of thylakoid protein phosphorylation at the substrate level: Reversible light-induced conformational changes expose the phosphorylation site of the light-harvesting complex II
Authors: Zer Hagit , Vink Martin , Keren Nir , Dilly-Hartwig Hans G. , Paulsen Harald , Herrmann Reinhold G. , Andersson Bertil , Ohad Itzhak
Published: July 2002

Light-dependent activation of thylakoid protein phosphorylation regulates the energy distribution between photosystems I and II of oxygen-evolving photosynthetic eukaryotes as well as the turnover of photosystem II proteins. So far the only known effect of light on the phosphorylation process is the redox-dependent regulation of the membrane-bound protein kinase(s) activity via plastoquinol bound to the cytochrome
bf
complex and the redox state of thylakoid dithiols. By using a partially purified thylakoid protein kinase and isolated native chlorophyll (chl)

a
/
b

light-harvesting complex II (LHCII), as well as recombinant LHCII, we find that illumination of the chl-protein substrate exposes the phosphorylation site to the kinase. Light does not activate the phosphorylation of the LHCII apoprotein nor the recombinant pigment-reconstituted complex lacking the N-terminal domain that contains the phosphothreonine site. The suggested light-induced conformational change exposing the N-terminal domain of LHCII to the kinase is evidenced also by an increase in its accessibility to tryptic cleavage after light exposure. Light activates preferentially the trimeric form of LHCII, and the process is paralleled by chl fluorescence quenching. Both phenomena are slowly reversible in darkness. Light-induced exposure of the LHCII N-terminal domain to the endogenous protein kinase(s) and tryptic cleavage occurs also in thylakoid membranes. These results demonstrate that light may regulate thylakoid protein phosphorylation not only via the signal transduction chain connecting redox reactions to the protein kinase activation, but also by affecting the conformation of the chl-protein substrate.
Genetic mapping of resistance to
Bacillus thuringiensis
toxins in diamondback moth using biphasic linkage analysis
Authors: Heckel David G. , Gahan Linda J. , Liu Yong-Biao , Tabashnik Bruce E.
Published: July 2002

Transgenic plants producing environmentally benign
Bacillus thuringiensis
(Bt) toxins are deployed increasingly for insect control, but their efficacy will be short-lived if pests adapt quickly. The diamondback moth (
Plutella xylostella
), a worldwide pest of vegetables, is the first insect to evolve resistance to Bt toxins in open-field populations. A recessive autosomal gene confers resistance to at least four Bt toxins and enables survival without adverse effects on transgenic plants. Allelic variants of this gene confer resistance in strains from Hawaii, Pennsylvania, and the Philippines. Here we exploited the biphasic nature of Lepidopteran genetic linkage to map this gene in diamondback moth with 207 amplified fragment length polymorphisms as DNA markers. We also cloned and sequenced an amplified fragment length polymorphism marker for the chromosome containing the Bt resistance gene. The results provide a powerful tool for facilitating progress in understanding, monitoring, and managing resistance to Bt.
Thiostrepton inhibits the turnover but not the GTPase of elongation factor G on the ribosome
Authors: Rodnina Marina V. , Savelsbergh Andreas , Matassova Natalia B. , Katunin Vladimir I. , Semenkov Yuri P. , Wintermeyer Wolfgang
Published: July 2002

The region around position 1067 in domain II of 23S rRNA frequently is referred to as the GTPase center of the ribosome. The notion is based on the observation that the binding of the antibiotic thiostrepton to this region inhibited GTP hydrolysis by elongation factor G (EF-G) on the ribosome at the conditions of multiple turnover. In the present work, we have reanalyzed the mechanism of action of thiostrepton. Results obtained by biochemical and fast kinetic techniques show that thiostrepton binding to the ribosome does not interfere with factor binding or with single-round GTP hydrolysis. Rather, the antibiotic inhibits the function of EF-G in subsequent steps, including release of inorganic phosphate from EF-G after GTP hydrolysis, tRNA translocation, and the dissociation of the factor from the ribosome, thereby inhibiting the turnover reaction. Structurally, thiostrepton interferes with EF-G footprints in the α-sarcin stem loop (A2660, A2662) located in domain VI of 23S rRNA. The results indicate that thiostrepton inhibits a structural transition of the 1067 region of 23S rRNA that is important for functions of EF-G after GTP hydrolysis.
Transgenic mice demonstrate AP-1 (activator protein-1) transactivation is required for tumor promotion
Authors: Young Matthew R. , Li Jian-Jian , Rincón Mercedes , Flavell Richard A. , Sathyanarayana B. K. , Hunziker Rosemarie , Colburn Nancy
Published: July 2002

Activator protein-1 (AP-1) is a transcription factor that consists of either a Jun-Jun homodimer or a Jun-Fos heterodimer. Transactivation of AP-1 is required for tumor promoter-induced transformation in mouse epidermal JB6 cells and for progression in mouse and human keratinocytes. Until now, the question of whether AP-1 transactivation is required for carcinogenesis
in vivo
has remained unanswered, as has the issue of functionally significant target genes. To address these issues we have generated a transgenic mouse in which transactivation mutant
c-jun
(TAM67), under the control of the human keratin-14 promoter, is expressed specifically in the basal cells of the epidermis where tumor induction is initiated. The keratin-14–TAM67 transgene was expressed in the epidermis, tongue, and cervix, with no apparent abnormalities in any tissue or organ. TAM67 expression blocked 12-
O
-tetradecanoylphorbol 13-acetate (TPA, phorbol 12-tetradecanoate 13-acetate) induction of the AP-1-regulated luciferase in AP-1 luciferase/TAM67 mice, but did not inhibit induction of candidate AP-1 target genes, collagenase-1 or stromelysin-3. More interestingly, TAM67 expression did not inhibit TPA-induced hyperproliferation. In two-stage skin carcinogenesis experiments, the transgenic animals showed a dramatic inhibition of papilloma induction. We conclude that transactivation of a subset of AP-1-dependent genes is required for tumor promotion and may be targeted for cancer prevention.
Altered dosage of the
Saccharomyces cerevisiae
spindle pole body duplication gene,
NDC1
, leads to aneuploidy and polyploidy
Authors: Chial H. J. , Giddings T. H. , Siewert E. A. , Hoyt M. A. , Winey M.
Published: July 2002

Saccharomyces cerevisiae
cells are exquisitely sensitive to altered dosage of the spindle pole body duplication gene,
NDC1
. We show that the
NDC1
locus is haploinsufficient because diploid yeast cells cannot survive with a single chromosomal copy of the
NDC1
gene. Diploid cells with a single copy of
NDC1
can survive by gaining an extra copy of the
NDC1
-containing chromosome.
NDC1
haploinsufficiency is a dominant loss-of-function phenotype that leads to aneuploidy. Furthermore, we report that overexpression of
NDC1
leads to spindle pole body duplication defects indistinguishable from those observed in
ndc1–1
mutant cells. Cells overexpressing
NDC1
arrest with monopolar spindles and exhibit increase-in-ploidy phenotypes. Thus, both increased and decreased
NDC1
dosage can lead to aneuploidy. The striking sensitivity of yeast cells to changes in
NDC1
gene dosage suggests a model for the behavior of some tumor suppressor genes and oncogenes in which loss-of-function mutations and overexpression, respectively, lead to increased genetic instability.
Decreased selective uptake of high density lipoprotein cholesteryl esters in apolipoprotein E knock-out mice
Authors: Arai Takeshi , Rinninger Franz , Varban Lee , Fairchild-Huntress Victoria , Liang Chien-Ping , Chen Wengen , Seo Toru , Deckelbaum Richard , Huszar Dennis , Tall Alan R.
Published: July 2002

Scavenger receptor BI (SR-BI) mediates the selective uptake of high density lipoprotein (HDL) cholesteryl esters (CE) by cells, i.e., the uptake of CE without degradation of HDL protein. Mice with attenuated expression of SR-BI, because of targeted gene mutation (SR-BIatt mice), have increased plasma HDL levels as a result of decreased selective uptake in the liver. To further evaluate the role of SR-BI in lipoprotein metabolism, compound apolipoprotein E knock-out (apoE0)/SR-BIatt mice were bred. Hepatic SR-BI protein was increased (2.3-fold) in apoE0 mice compared with wild type (wt) and was reduced significantly in apoE0/SR-BIatt mice. However, the plasma lipoprotein profile of apoE0 and apoE0/SR-BIatt mice was identical. This was explained by HDL turnover studies that revealed that the selective clearance of HDL CE by the liver and adrenal was already profoundly impaired in apoE0 mice compared with wt (28% of wt in liver). A similar decrease in selective uptake was seen when apoE0 HDL was incubated with isolated apoE0 hepatocytes. The results suggest that apoE plays a major role in the selective clearance of HDL CE by the liver and adrenal gland, possibly by facilitating the presentation of HDL to SR-BI at the cell surface.
Pressure-enhanced ortho-para conversion in solid hydrogen up to 58 GPa
Authors: Eggert Jon H. , Karmon Eran , Hemley Russell J. , Mao Ho-kwang , Goncharov Alexander F.
Published: July 2002

We measured the ortho-para conversion rate in solid hydrogen by using Raman scattering in a diamond-anvil cell, extending previous measurements by a factor of 60 in pressure. We confirm previous experiments that suggested a decrease in the conversion rate above about 0.5 GPa. We observe a distinct minimum at 3 GPa followed by a drastic increase in the conversion rate to our maximum pressure of 58 GPa. This pressure enhancement of conversion is not predicted by previous theoretical treatments and must be due to a new conversion pathway.
Isolation of a Chinese hamster ovary cell mutant defective in intramitochondrial transport of phosphatidylserine
Authors: Emoto Kazuo , Kuge Osamu , Nishijima Masahiro , Umeda Masato
Published: July 2002

A CHO-K1 cell mutant with a specific decrease in cellular phosphatidylethanolamine (PE) level was isolated as a variant resistant to Ro09–0198, a PE-directed antibiotic peptide. The mutant was defective in the phosphatidylserine (PS) decarboxylation pathway for PE formation, in which PS produced in the endoplasmic reticulum is transported to mitochondria and then decarboxylated by an inner mitochondrial membrane enzyme, PS decarboxylase. Neither PS formation nor PS decarboxylase activity was reduced in the mutant, implying that the mutant is defective in some step of PS transport. The transport processes of phospholipids between the outer and inner mitochondrial membrane were analyzed by use of isolated mitochondria and two fluorescence-labeled phospholipid analogs, 1-palmitoyl-2-{
N
-[6(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]caproyl}-PS (C6-NBD-PS) and C6-NBD-phosphatidylcholine (C6-NBD-PC). On incubation with the CHO-K1 mitochondria, C6-NBD-PS was readily decarboxylated to C6-NBD-PE, suggesting that the PS analog was partitioned into the outer leaflet of mitochondria and then translocated to the inner mitochondrial membrane. The rate of decarboxylation of C6-NBD-PS in the mutant mitochondria was reduced to ≈40% of that in the CHO-K1 mitochondria. The quantity of phospholipid analogs translocated from the outer leaflet of mitochondria into inner mitochondrial membranes was further examined by selective extraction of the analogs from the outer leaflet of mitochondria. In the mutant mitochondria, the translocation of C6-NBD-PS was significantly reduced, whereas the translocation of C6-NBD-PC was not affected. These results indicate that the mutant is defective in PS transport between the outer and inner mitochondrial membrane and provide genetic evidence for the existence of a specific mechanism for intramitochondrial transport of PS.
Chromatin modification by DNA tracking
Authors: Travers Andrew
Published: July 2002

In general, the transcriptional competence of a chromatin domain is correlated with increased sensitivity to DNase I cleavage. A recent observation that actively transcribing RNA polymerase II piggybacks a histone acetyltranferase activity [Wittschieben, B., Otero, G., de Bizemont, T., Fellows, J., Erdjument-Bromage, H., Ohba, R., Li, Y., Allis, C. D., Tempst, P. & Svejstrup, J. Q. (1999)
Mol. Cell
4, 123–128] implies that the state of histone acetylation, and hence the ability of chromatin to fold, can be altered by a processive mechanism. In this article, it is proposed that tracking-mediated chromatin modification could create and/or maintain an open configuration in a complete chromatin domain including both intra- and extragenic regions. This mechanism suggests a putative functional role for the extragenic transcription observed at the β-globin and other loci in vertebrate cells.
Trafficking of spontaneously endocytosed MHC proteins
Authors: Chiu Isaac , Davis Daniel M. , Strominger Jack L.
Published: July 2002

Class I MHC protein primarily presents endogenous antigen but also may present exogenous antigen. Here, we investigated the intracellular pathway of spontaneously internalized class I MHC protein by confocal microscopy. β2-microglobulin (β2m), labeled with a single fluorophore, was exchanged at the surface of B cell transfectants to specifically mark cell surface and endocytosed class I MHC protein. Intracellular β2m colocalized with fluorophore-conjugated transferrin, implying that class I MHC protein endocytosed into early endosomes. These endosomes containing fluorescent β2m were found close to or within the Golgi apparatus, marked by fluorescent ceramide. Even after 24 hr of incubation, very little fluorescent β2m was found in intracellular organelles stained by DiOC6, marking the endoplasmic reticulum, or fluorophore-conjugated low density lipoprotein, marking late endosomes and lysosomes. Fluorophore-conjugated superantigens (staphylococcal enterotoxin A and B), presumed to enter cells bound to class II MHC protein, also were found to endocytose into β2m-containing early endosomes. Staining with mAb and use of transfectants expressing MHC protein attached to green fluorescent protein confirmed the presence of intracellular compartments rich in both class I and II MHC protein and demonstrated that class I and II MHC protein also colocalize in discrete microdomains at the cell surface. These cell surface microdomains also contained transferrin receptor and often were juxtaposed to cholesterol-rich lipid rafts. Thus, class I and II MHC protein meet in microdomains of the plasma membrane and endocytose into early endosomes, where both may acquire and present exogenous antigen.
Nuclear tRNA aminoacylation and its role in nuclear export of endogenous tRNAs in
Saccharomyces
cerevisiae
Authors: Sarkar Srimonti , Azad Abul K. , Hopper Anita K.
Published: July 2002

Nuclear tRNA aminoacylation was proposed to provide a proofreading step in
Xenopus
oocytes, ensuring nuclear export of functional tRNAs [Lund, E. & Dahlberg, J. E. (1998)
Science
282, 2082–2085]. Herein, it is documented that tRNA aminoacylation also occurs in yeast nuclei and is important for tRNA export. We propose that tRNA aminoacylation functions in one of at least two parallel paths of tRNA export in yeast. Alteration of one aminoacyl-tRNA synthetase affects export of only cognate tRNA, whereas alterations of two other aminoacyl-tRNA synthetases affect export of both cognate and noncognate tRNAs. Saturation of tRNA export pathway is a possible explanation of this phenomenon.
A bcr-3 isoform of RARα-PML potentiates the development of PML-RARα-driven acute promyelocytic leukemia
Authors: Pollock Jessica L. , Westervelt Peter , Kurichety Arun K. , Pelicci Pier Giuseppe , Grisolano Jay L. , Ley Timothy J.
Published: July 2002

Acute promyelocytic leukemia (APML) most often is associated with the balanced reciprocal translocation t(15;17) (q22;q11.2) and the expression of both the PML-RARα and RARα-PML fusion cDNAs that are formed by this translocation. In this report, we investigated the biological role of a bcr-3 isoform of RARα-PML for the development of APML in a transgenic mouse model. Expression of RARα-PML alone in the early myeloid cells of transgenic mice did not alter myeloid development or cause APML, but its expression significantly increased the penetrance of APML in mice expressing a bcr-1 isoform of PML-RARα (15% of animals developed APML with PML-RARα alone vs. 57% with both transgenes,P< 0.001). The latency of APML development was not altered substantially by the expression of RARα-PML, suggesting that it does not behave as a classical “second hit” for development of the disease. Leukemias that arose from doubly transgenic mice were less mature than those from PML-RARα transgenic mice, but they both responded to all-transretinoic acidin vitro. These findings suggest that PML-RARα drives the development of APML and defines its basic phenotype, whereas RARα-PML potentiates this phenotype via mechanisms that are not yet understood.
Autoinducer binding by the quorum-sensing regulator TraR increases affinity for target promoters
in vitro
and decreases TraR turnover rates in whole cells
Authors: Zhu Jun , Winans Stephen C.
Published: July 2002

TraR is an
Agrobacterium
transcriptional regulator whose activity requires the pheromone
N
-3-oxooctanoyl-
l
-homoserine lactone. TraR was purified as a complex with the pheromone and contained one pheromone molecule per protein monomer. TraR–pheromone complexes bound to a single DNA site and activated two promoters that flank this site. Promoter expression was elevated 30-fold by using a supercoiled template. Pheromone binding increased the affinity of TraR for this binding site. Pheromone also increased TraR abundance
in vivo
by causing a 20-fold decrease in TraR turnover rates.
Continuous
in vitro
propagation and differentiation of cultures of the intramolluscan stages of the human parasite
Schistosoma mansoni
Authors: Ivanchenko Maria G. , Lerner Jennifer P. , McCormick Renee S. , Toumadje Arazdordi , Allen Brady , Fischer Kay , Hedstrom Olaf , Helmrich Angela , Barnes David W. , Bayne Christopher J.
Published: July 2002

The metazoan parasitic blood flukes,
Schistosoma
spp., infect over 200 million people worldwide and cause extensive human morbidity and mortality. Research strategies for development of anti-schistosomal agents are impeded by the organism’s complex molluscan–mammalian life cycle, which limits experimental approaches and availability of material. We derived long-term continuously proliferative cultures of
Schistosoma mansoni
sporocysts capable of generating cercariae
in vitro
. Cultured organisms retained the ability to parasitize the host, and they exhibited developmental regulation of candidate stage-specific genes in the host-free culture system. Evidence for expression of a reverse transcriptase also was found in the cultured organisms, pointing to this activity as a possible mechanistic contributor to the dynamic relationship between the parasite and its hosts. Continuous
in vitro
propagation of the asexual sporocyst stage allows isolation of clonally derived parasite populations and provides a means to study schistosomal molecular genetics, metabolism, and evasion of host defenses.
Rescue of dystrophin expression in
mdx
mouse muscle by RNA/DNA oligonucleotides
Authors: Rando Thomas A. , Disatnik Marie-Helene , Zhou Lucy Z.-H.
Published: July 2002

Chimeric RNA/DNA oligonucleotides (“chimeraplasts”) have been shown to induce single base alterations in genomic DNA both
in vitro
and
in vivo
. The
mdx
mouse strain has a point mutation in the dystrophin gene, the consequence of which is a muscular dystrophy resulting from deficiency of the dystrophin protein in skeletal muscle. To test the feasibility of chimeraplast-mediated gene therapy for muscular dystrophies, we used a chimeraplast (designated “MDX1”) designed to correct the point mutation in the dystrophin gene in
mdx
mice. After direct injection of MDX1 into muscles of
mdx
mice, immunohistochemical analysis revealed dystrophin-positive fibers clustered around the injection site. Two weeks after single injections into tibialis anterior muscles, the maximum number of dystrophin-positive fibers (approximately 30) in any muscle represented 1–2% of the total number of fibers in that muscle. Ten weeks after single injections, the range of the number of dystrophin-positive fibers was similar to that seen after 2 wk, suggesting that the expression was stable, as would be predicted for a gene-conversion event. Staining with exon-specific antibodies showed that none of these were “revertant fibers.” Furthermore, dystrophin from MDX1-injected muscles was full length by immunoblot analysis. No dystrophin was detectable by immunohistochemical or immunoblot analysis after control chimeraplast injections. Finally, reverse transcription–PCR analysis demonstrated the presence of transcripts with the wild-type dystrophin sequence only in
mdx
muscles injected with MDX1 chimeraplasts. These results provide the foundation for further studies of chimeraplast-mediated gene therapy as a therapeutic approach to muscular dystrophies and other genetic disorders of muscle.
Calsenilin reverses presenilin-mediated enhancement of calcium signaling
Authors: Leissring Malcolm A. , Yamasaki Tritia R. , Wasco Wilma , Buxbaum Joseph D. , Parker Ian , LaFerla Frank M.
Published: July 2002

Most cases of autosomal-dominant familial Alzheimer's disease are linked to mutations in the presenilin genes (
PS1
and
PS2
). In addition to modulating β-amyloid production, presenilin mutations also produce highly specific and selective alterations in intracellular calcium signaling. Although the molecular mechanisms underlying these changes are not known, one candidate molecular mediator is calsenilin, a recently identified calcium-binding protein that associates with the C terminus of both PS1 and PS2. In this study, we investigated the effects of calsenilin on calcium signaling in
Xenopus
oocytes expressing either wild-type or mutant PS1. In this system, mutant PS1 potentiated the amplitude of calcium signals evoked by inositol 1,4,5-trisphosphate and also accelerated their rates of decay. We report that calsenilin coexpression reverses both of these potentially pathogenic effects. Notably, expression of calsenilin alone had no discernable effects on calcium signaling, suggesting that calsenilin modulates these signals by a mechanism independent of simple calcium buffering. Our findings further suggest that the effects of presenilin mutations on calcium signaling are likely mediated through the C-terminal domain, a region that has also been implicated in the modulation of β-amyloid production and cell death.
CXR, a chicken xenobiotic-sensing orphan nuclear receptor, is related to both mammalian pregnane X receptor (PXR) and constitutive androstane receptor (CAR)
Authors: Handschin Christoph , Podvinec Michael , Meyer Urs A.
Published: July 2002

Nuclear receptors constitute a large family of ligand-modulated
transcription factors that mediate cellular responses to small
lipophilic molecules, including steroids, retinoids, fatty acids, and
exogenous ligands. Orphan nuclear receptors with no known
endogenous ligands have been discovered to regulate
drug-mediated induction of cytochromes P450 (CYP), the major
drug-metabolizing enzymes. Here, we report the cloning of an orphan
nuclear receptor from chicken, termed chicken xenobiotic receptor
(CXR), that is closely related to two mammalian
xenobiotic-activated receptors, the pregnane X receptor (PXR) and
the constitutive androstane receptor (CAR). Expression of CXR is
restricted to tissues where drug induction of CYPs predominantly
occurs, namely liver, kidney, small intestine, and colon. Furthermore,
CXR binds to a previously identified phenobarbital-responsive enhancer
unit (PBRU) in the 5′-flanking region of the chicken
CYP2H1
gene. A variety of drugs, steroids, and chemicals
activate CXR in CV-1 monkey cell transactivation assays. The same
agents induce PBRU-dependent reporter gene expression and
CYP2H1
transcription in a chicken hepatoma cell line.
These results provide convincing evidence for a major role of CXR in
the regulation of
CYP2H1
and add a member to the family
of xenobiotic-activated orphan nuclear receptors.
Antibodies have the intrinsic capacity to destroy antigens
Authors: Wentworth Anita D. , Jones Lyn H. , Wentworth Paul , Janda Kim D. , Lerner Richard A.
Published: July 2002

Research throughout the last century has led to a consensus as to the strategy of the humoral component of the immune system. The essence is that, for killing, the antibody molecule activates additional systems that respond to antibody–antigen union. We now report that the immune system seems to have a previously unrecognized chemical potential intrinsic to the antibody molecule itself. All antibodies studied, regardless of source or antigenic specificity, can convert molecular oxygen into hydrogen peroxide, thereby potentially aligning recognition and killing within the same molecule. Aside from pointing to a new chemical arm for the immune system, these results may be important to the understanding of how antibodies evolved and what role they may play in human diseases.
Optical probing of neuronal circuits with calcium indicators
Authors: Peterlin Zita A. , Kozloski James , Mao Bu-Qing , Tsiola Areti , Yuste Rafael
Published: July 2002

An experimental difficulty in unraveling circuits in the mammalian nervous system is the identification of postsynaptic targets of a given neuron. Besides ultrastructural reconstructions, simultaneous recordings from pairs of cells in brain slices have been used to identify connected neurons. We describe in this paper a technique using calcium imaging that allows rapid identification of potential postsynaptic targets. This method consists of stimulating one neuron (“trigger”) while imaging a population of cells to detect which other neurons (“followers”) are activated by the trigger. By using bulk-loading of calcium indicators in slices of mouse visual cortex, we demonstrate that neurons that display somatic calcium transients time-locked to the spikes of a trigger neuron can be monosynaptically connected to it. This technique could be applied to reconstruct and assay circuits in the central nervous system.
A statistical mechanical method to optimize energy functions for protein folding
Authors: Bastolla Ugo , Vendruscolo Michele , Knapp Ernst-Walter
Published: July 2002

We present a method for deriving energy functions for protein folding by maximizing the thermodynamic average of the overlap with the native state. The method has been tested by using the pairwise contact approximation of the energy function and generating alternative structures by threading sequences over a database of 1,169 structures. With the derived energy function, most native structures: (
i
) have minimal energy and (
ii
) are thermodynamically rather stable, and (
iii
) the corresponding energy landscapes are smooth. Precisely, 92% of the 1,013 x-ray structures are stabilized. Most failures can be attributed to the neglect of interactions between chains forming polychain proteins and of interactions with cofactors. When these are considered, only nine cases remain unexplained. In contrast, 38% of NMR structures are not assigned properly.
Prolyl 4-hydroxylase is required for viability and morphogenesis in
Caenorhabditis elegans
Authors: Friedman Lisa , Higgin Joshua J. , Moulder Gary , Barstead Robert , Raines Ronald T. , Kimble Judith
Published: July 2002

The genome of
Caenorhabditis
elegans
possesses two genes,
dpy-18
and
phy-2,
that encode α subunits of the enzyme prolyl
4-hydroxylase. We have generated deletions within each gene to
eliminate prolyl 4-hydroxylase activity from the animal. The
dpy-18
mutant has an aberrant body morphology,
consistent with a role of prolyl 4-hydroxylase in formation of the body
cuticle. The
phy-2
mutant is phenotypically wild type.
However, the
dpy-18; phy-2
double mutant is not viable,
suggesting an essential role for prolyl 4-hydroxylase that is normally
accomplished by either
dpy-18
or
phy-2
.
The effects of the double mutation were mimicked by small-molecule
inhibitors of prolyl 4-hydroxylase, validating the genetic results and
suggesting that
C. elegans
can serve as a model system
for the discovery of new inhibitors.
Improved DNA binding specificity from polyzinc finger peptides by using strings of two-finger units
Authors: Moore Michael , Klug Aaron , Choo Yen
Published: July 2002

Multizinc finger peptides are likely to reach increased prominence
in the search for the “ideal” designer transcription factor for
in vivo
applications such as gene therapy. However, for
these treatments to be effective and safe, the peptides must bind with
high affinity and, more importantly, with great specificity. Our
previous research has shown that zinc finger arrays can be made to bind
18 bp of DNA with picomolar affinity, but also has suggested that
arrays of fingers also may bind tightly to related sequences. This work
addresses the question of zinc finger DNA binding specificity. We show
that by changing the way in which zinc finger arrays are
constructed—by linking three two-finger domains rather than two
three-finger units—far greater target specificity can be achieved
through increased discrimination against mutated or closely related
sequences. These new peptides have the added capability of being able
to span two short gaps of unbound DNA, although still binding with
picomolar affinity to their target sites. We believe that this new
method of constructing zinc finger arrays will offer greater efficacy
in the fields of gene therapy and in the production of transgenic
organisms than previously reported zinc finger arrays.
Eotaxin in serum of patients with asthma or chronic obstructive pulmonary disease: relationship with eosinophil cationic protein and lung function
Authors: Jahnz-Ró̀zyk Karina , Plusa Tadeusz , Mierzejewska Justyna
Published: July 2002

This study was undertaken to investigate the correlation between the serum ECP and the serum eotaxin level, and disease activity as evaluated with pulmonary function in patients with asthma or chronic obstructive pulmonary disease (COPD). 20 patients with stable asthma and 15 patients with COPD, and 15 subjects of the control group took part in this study. The analysis of ECP was performed according to the manufacturer’s directions (Pharmacia Diagnostics AB, Uppsala, Sweden). The ELISA test was used to measure eotaxin levels in sserum (kits from R&D, USA). The levels of ECP were
16.9 ± 6.3 μg/L in patients with asthma, 15.1 ± 9.3 μg/L in patients with COPD and
11.8 ± 6.2 μg/L in the control group (p < 0.05). There was no significant difference in the asthma ECP level compared with the ECP level in COPD. There was a significant difference between the ECP plasma level in asthma compared with the ECP plasma level in the control group (p < 0.05). The levels of eotaxin were 175.8 ± 49.3 pg/mL in the control group. There was a correlation of ECP and the eotaxin level in asthma patients (r = +0.5, p < 0.05). The percentage fall in FEV1 correlated with eotaxin level in asthma
(r = −0.3, p < 0.05) and with the eotaxin level in COPD (r = −0.05, p < 0.05). Serum outcomes of eotaxin and ECP levels appear to be a useful indicator of atopic asthma, and might provide complementary data disease monitoring. Therefore, further investigations are required to clarify whether serum eotaxin measurements have a role in the clinical evaluation in COPD.