bluuebunny/crossref_metadata_2025 huggingface dataset:
Mailborne transmission of anthrax: Modeling and implications
Authors: Webb Glenn F. , Blaser Martin J.
Published: July 2002

A mathematical model is developed to analyze the transmission of inhalational anthrax through the postal system by cross-contamination of mail. The model consists of state vectors describing the numbers of cross-contaminated letters generated, the numbers of anthrax spores on these letters, the numbers of resulting infections in recipients, and matrices of transition probabilities acting on these vectors. The model simulates the recent outbreak in the United States, and provides a general framework to investigate the potential impact of possible future outbreaks.
Structural features of an influenza virus promoter and their implications for viral RNA synthesis
Authors: Bae Sung-Hun , Cheong Hae-Kap , Lee Joon-Hwa , Cheong Chaejoon , Kainosho Masatsune , Choi Byong-Seok
Published: July 2002

The influenza A virus, a severe pandemic pathogen, has a segmented RNA genome consisting of eight single-stranded RNA molecules. The 5′ and 3′ ends of each RNA segment recognized by the influenza A virus RNA-dependent RNA polymerase direct both transcription and replication of the virus's RNA genome. Promoter binding by the viral RNA polymerase and formation of an active open complex are prerequisites for viral replication and proliferation. Here we describe the solution structure of this promoter as solved by multidimensional, heteronuclear magnetic resonance spectroscopy. Our studies show that the viral promoter has a significant dynamic nature and reveal an unusual displacement of an adenosine that forms a novel (A-A)⋅U motif and a C-A mismatch stacked in a helix. The characterized structural features of the promoter imply that the specificity of polymerase binding results from an internal RNA loop. In addition, an unexpected bending (46 ± 10°) near the initiation site suggests the existence of a promoter recognition mechanism similar to that of DNA-dependent RNA polymerase and a possible regulatory function for the terminal structure during open complex formation.
The cloned rat pancreatic polypeptide receptor exhibits profound differences to the orthologous receptor.
Authors: Lundell I , Statnick M A , Johnson D , Schober D A , Starbäck P , Gehlert D R , Larhammar D
Published: July 2002

Pancreatic polypeptide (PP) is produced in the islets of Langerhans and released in response to meals. It belongs to a family of peptides that also includes neuropeptide Y and peptide YY. In the present communication, we describe a rat receptor with high affinity for PP, therefore named PP1. Clones for the PP1 receptor were obtained by PCR using sequence information for the neuropeptide Y receptor Y1 from several species. The PP1 receptor has 46% overall amino acid sequence identity to the rat Y1 receptor and 56% identity in the transmembrane regions. The PP1 receptor displays a pharmacological profile that is distinct from previously described neuropeptide Y-family receptors. In competition with iodinated bovine PP, it binds rat PP with an affinity (K(i)) of 0.017 nM, while the affinities for peptide YY and neuropeptide Y are substantially lower with K(i) values of 162 and 192 nM, respectively. In stably transfected CHO cells, the PP1 receptor inhibits forskolin-stimulated cAMP synthesis. Northern blot hybridizations to a panel of mRNAs detected transcripts in testis and lung. A faint band was seen in colon and total brain. In contrast, the human receptor is expressed primarily in colon and small intestine. Whereas rat and human PP1 bind PP with the same affinity, the rat receptor has much lower affinity than its human ortholog for peptide YY and neuropeptide Y. Interestingly, the amino acid sequence identity between rat and human PP1 is only 75%. Thus, the sequence, the tissue distribution, and the binding profile of the PP1 receptor differ considerably between rat and human.
Cloning and expression in Escherichia coli of the OGG1 gene of Saccharomyces cerevisiae, which codes for a DNA glycosylase that excises 7,8-dihydro-8-oxoguanine and 2,6-diamino-4-hydroxy-5-N-methylformamidopyrimidine.
Authors: van der Kemp P A , Thomas D , Barbey R , de Oliveira R , Boiteux S
Published: July 2002

A spontaneous mutator strain of Escherichia coli (fpg mutY) was used to clone the OGG1 gene of Saccharomyces cerevisiae, which encodes a DNA glycosylase activity that excises 7,8-dihydro-8-oxoguanine (8-OxoG). E. coli (fpg mutY) was transformed by a yeast DNA library, and clones that showed a reduced spontaneous mutagenesis were selected. The antimutator activity was associated with pYSB10, an 11-kbp recombinant plasmid. Cell-free extracts of E. coli (fpg mutY) harboring pYSB10 possess an enzymatic activity that cleaves a 34-mer oligonucleotide containing a single 8-oxoG opposite a cytosine (8-OxoG/C). The yeast DNA fragment of 1.7 kbp that suppresses spontaneous mutagenesis and overproduces the 8-OxoG/C cleavage activity was sequenced and mapped to chromosome XIII. DNA sequencing identified an open reading frame, designated OGG1, which encodes a protein of 376 amino acids with a molecular mass of 43 kDa. The OGG1 gene was inserted in plasmid pUC19, yielding pYSB110. E. coli (fpg) harboring pYSB110 was used to purify the Ogg1 protein of S. cerevisiae to apparent homogeneity. The Ogg1 protein possesses a DNA glycosylase activity that releases 8-OxoG and 2,6-diamino-4-hydroxy-5-N-methylformamidopyrimidine. The Ogg1 protein preferentially incises DNA that contains 8-OxoG opposite cytosine (8-OxoG/C) or thymine (8-OxoG/T). In contrast, Ogg1 protein does not incise the duplex where an adenine is placed opposite 8-OxoG (8-OxoG/A). The mechanism of strand cleavage by Ogg1 protein is probably due to the excision of 8-OxoG followed by a beta-elimination at the resulting apurinic/apyrimidinic site.
Congruence of fatty acid methyl ester profiles and morphological characters of arbuscular mycorrhizal fungi in Gigasporaceae.
Authors: Bentivenga S P , Morton J B
Published: July 2002

Arbuscular mycorrhizal (AM) fungi (Order Glomales, Class Zygomycetes) are a diverse group of soil fungi that form mutualistic associations with the roots of most species of higher plants. Despite intensive study over the past 25 years, the phylogenetic relationships among AM fungi, and thus many details of evolution of the symbiosis, remain unclear. Cladistic analysis was performed on fatty acid methyl ester (FAME) profiles of 15 species in Gigaspora and Scutellospora (family Gigasporaceae) by using a restricted maximum likelihood approach of continuous character data. Results were compared to a parsimony analysis of spore morphological characters of the same species. Only one tree was generated from each character set. Morphological and developmental data suggest that species with the simplest spore types are ancestral whereas those with complicated inner wall structures are derived. Spores of those species having a complex wall structure pass through stages of development identical to the mature stages of simpler spores, suggesting a pattern of classical Haeckelian recapitulation in evolution of spore characters. Analysis of FAME profiles supported this hypothesis when Glomus leptotichum was used as the outgroup. However, when Glomus etunicatum was chosen as the outgroup, the polarity of the entire tree was reversed. Our results suggest that FAME profiles contain useful information and provide independent criteria for generating phylogenetic hypotheses in AM fungi. The maximum likelihood approach to analyzing FAME profiles also may prove useful for many other groups of organisms in which profiles are empirically shown to be stable and heritable.
A single mutation that increases maize seed weight.
Authors: Giroux M J , Shaw J , Barry G , Cobb B G , Greene T , Okita T , Hannah L C
Published: July 2002

The maize endosperm-specific gene shrunken2 (Sh2) encodes the large subunit of the heterotetrameric starch synthetic enzyme adenosine diphosphoglucose pyrophosphorylase (AGP; EC 2.7.7.27). Here we exploit an in vivo, site-specific mutagenesis system to create short insertion mutations in a region of the gene known to be involved in the allosteric regulation of AGP. The site-specific mutagen is the transposable element dissociation (Ds). Approximately one-third (8 of 23) of the germinal revertants sequenced restored the wild-type sequence, whereas the remaining revertants contained insertions of 3 or 6 bp. All revertants retained the original reading frame 3' to the insertion site and involved the addition of tyrosine and/or serine. Each insertion revertant reduced total AGP activity and the amount of the SH2 protein. The revertant containing additional tyrosine and serine residues increased seed weight 11-18% without increasing or decreasing the percentage of starch. Other insertion revertants lacking an additional serine reduced seed weight. Reduced sensitivity to phosphate, a long-known inhibitor of AGP, was found in the high seed-weight revertant. This alteration is likely universally important since insertion of tyrosine and serine in the potato large subunit of AGP at the comparable position and expression in Escherichia coli also led to a phosphate-insensitive enzyme. These results show that single gene mutations giving rise to increased seed weight, and therefore perhaps yield, are clearly possible in a plant with a long history of intensive and successful breeding efforts.
Hepatitis B virus transactivator protein, HBx, associates with the components of TFIIH and stimulates the DNA helicase activity of TFIIH.
Authors: Qadri I , Conaway J W , Conaway R C , Schaack J , Siddiqui A
Published: July 2002

Human hepatitis B virus genome encodes a protein, termed HBx, that is widely recognized as a transcriptional transactivator. While HBx does not directly bind cis-acting transcriptional control elements, it has been shown to associate with cellular proteins that bind DNA. Because HBx transactivated a large number of viral/cellular transcriptional control elements, we looked for its targets within the components of the basal transcriptional machinery. This search led to the identification of its interactions with TFIIH. Here, we show that HBx interacts with yeast and mammalian TFIIH complexes both in vitro and in vivo. These interactions between HBx and the components of TFIIH are supported by several lines of evidence including results from immunoprocedures and direct methods of measuring interactions. We have identified ERCC3 and ERCC2 DNA helicase subunits of holoenzyme TFIIH as targets of HBx interactions. Furthermore, the DNA helicase activity of purified TFIIH from rat liver and, individually, the ERCC2 component of TFIIH is stimulated in the presence of HBx. These observations suggest a role for HBx in transcription and DNA repair.
Interactions between two divalent ion binding sites in
N
-methyl-
d
-aspartate receptor channels
Authors: Sharma Geeta , Stevens Charles F.
Published: July 2002

N
-methyl-
d
-aspartate receptor channels
exhibit a high permeability for calcium ions. In this report, we
confirm that calcium ions permeate effectively through the wild-type
channels, and find that their presence within the pore blocks the flux
of sodium and other ions. Further proof for this ionic block comes from
the analysis of the ɛ1(N614Q) mutation where the high permeability of
calcium is unchanged but the block by calcium ions is increased
twofold. In both the wild-type and mutant channels, calcium ion block
is independent of membrane voltage; therefore, the calcium binding site
is outside the voltage gradient through the pore and must be close to
the extracellular mouth of the ion conductance pathway. This calcium
site is distinct from the magnesium binding site, which lies 80% into
the pore’s electrostatic field and thus exhibits a marked voltage
dependence of binding. The ɛ1(N614Q) mutation reduces the affinity of
magnesium ion for its binding site but increases the affinity of
calcium ion for its binding site. Since a single mutation perturbs two
distinct binding sites in opposite ways, we postulate that binding of
divalent ions at the two sites interact.
Redesign of soluble fatty acid desaturases from plants for altered substrate specificity and double bond position
Authors: Cahoon Edgar B. , Lindqvist Ylva , Schneider Gunter , Shanklin John
Published: July 2002

Acyl-acyl carrier protein (ACP) desaturases introduce double bonds at specific positions in fatty acids of defined chain lengths and are one of the major determinants of the monounsaturated fatty acid composition of vegetable oils. Mutagenesis studies were conducted to determine the structural basis for the substrate and double bond positional specificities displayed by acyl-ACP desaturases. By replacement of specific amino acid residues in a Δ
6
-palmitoyl (16:0)-ACP desaturase with their equivalents from a Δ
9
-stearoyl (18:0)-ACP desaturase, mutant enzymes were identified that have altered fatty acid chain-length specificities or that can insert double bonds into either the Δ
6
or Δ
9
positions of 16:0- and 18:0-ACP. Most notably, by replacement of five amino acids (A181T/A200F/S205N/L206T/G207A), the Δ
6
-16:0-ACP desaturase was converted into an enzyme that functions principally as a Δ
9
-18:0-ACP desaturase. Many of the determinants of fatty acid chain-length specificity in these mutants are found in residues that line the substrate binding channel as revealed by x-ray crystallography of the Δ
9
-18:0-ACP desaturase. The crystallographic model of the active site is also consistent with the diverged activities associated with naturally occurring variant acyl-ACP desaturases. In addition, on the basis of the active-site model, a Δ
9
-18:0-ACP desaturase was converted into an enzyme with substrate preference for 16:0-ACP by replacement of two residues (L118F/P179I). These results demonstrate the ability to rationally modify acyl-ACP desaturase activities through site-directed mutagenesis and represent a first step toward the design of acyl-ACP desaturases for the production of novel monounsaturated fatty acids in transgenic oilseed crops.
Interaction between replication protein A and p53 is disrupted after UV damage in a DNA repair-dependent manner
Authors: Abramova Natalia A. , Russell Jackie , Botchan Michael , Li Rong
Published: July 2002

Replication protein A (RPA) is required for both DNA replication and nucleotide excision repair. Previous studies have shown that RPA interacts with the tumor suppressor p53. Herein, we have mapped a 20-amino acid region in the N-terminal part of p53 that is essential for its binding to RPA. This region is distinct from the minimal activation domain of p53 previously identified. We also demonstrate that UV radiation of cells greatly reduces the ability of RPA to bind to p53. Interestingly, damage-induced hyperphosphorylated RPA does not associate with p53. Furthermore, down-regulation of the RPA/p53 interaction is dependent upon the capability of cells to perform global genome repair. On the basis of these data, we propose that RPA may participate in the coordination of DNA repair with the p53-dependent checkpoint control by sensing UV damage and releasing p53 to activate its downstream targets.
Molecular cloning and functional expression of a human cDNA encoding the antimutator enzyme 8-hydroxyguanine-DNA glycosylase
Authors: Roldán-Arjona Teresa , Wei Ying-Fei , Carter Kenneth C. , Klungland Arne , Anselmino Catherine , Wang Rui-Ping , Augustus Meena , Lindahl Tomas
Published: July 2002

The major mutagenic base lesion in DNA caused by exposure to reactive oxygen species is 8-hydroxyguanine (8-oxo-7,8-dihydroguanine). In bacteria and
Saccharomyces cerevisiae
, this damaged base is excised by a DNA glycosylase with an associated lyase activity for chain cleavage. We have cloned, sequenced, and expressed a human cDNA with partial sequence homology to the relevant yeast gene. The encoded 47-kDa human enzyme releases free 8-hydroxyguanine from oxidized DNA and introduces a chain break in a double-stranded oligonucleotide specifically at an 8-hydroxyguanine residue base paired with cytosine. Expression of the human protein in a DNA repair-deficient
E. coli mutM mutY
strain partly suppresses its spontaneous mutator phenotype. The gene encoding the human enzyme maps to chromosome 3p25. These results show that human cells have an enzyme that can initiate base excision repair at mutagenic DNA lesions caused by active oxygen.
Adjoint modular Galois representations and their Selmer groups
Authors: Hida Haruzo , Tilouine Jacques , Urban Eric
Published: July 2002

In the last 15 years, many class number formulas and main
conjectures have been proven. Here, we discuss such formulas on the
Selmer groups of the three-dimensional adjoint representation ad(φ)
of a two-dimensional modular Galois representation φ. We start with
the
p
-adic Galois representation φ
0
of a
modular elliptic curve
E
and present a formula expressing
in terms of
L
(1, ad(φ
0
)) the intersection
number of the elliptic curve
E
and the complementary
abelian variety inside the Jacobian of the modular curve. Then we
explain how one can deduce a formula for the order of the Selmer group
Sel(ad(φ
0
)) from the proof of Wiles of the
Shimura–Taniyama conjecture. After that, we generalize the formula in
an Iwasawa theoretic setting of one and two variables. Here the first
variable,
T
, is the weight variable of the universal
p
-ordinary Hecke algebra, and the second variable is the
cyclotomic variable
S
. In the one-variable case, we let φ
denote the
p
-ordinary Galois representation with values in
GL
2
(Z

p

[[
T
]])
lifting φ
0
, and the characteristic power series of the
Selmer group Sel(ad(φ)) is given by a
p
-adic
L
-function interpolating
L
(1,
ad(φ

k

)) for weight
k
+ 2
specialization φ

k

of φ. In the
two-variable case, we state a main conjecture on the characteristic
power series in Z

p

[[
T
,
S
]] of Sel(ad(φ) ⊗ ν
−1
), where ν is
the universal cyclotomic character with values in
Z

p

[[
S
]]. Finally, we
describe our recent results toward the proof of the conjecture and a
possible strategy of proving the main conjecture using
p
-adic Siegel modular forms.
An artificial cytochrome P450 that hydroxylates unactivated carbons with regio- and stereoselectivity and useful catalytic turnovers
Authors: Breslow Ronald , Huang Ying , Zhang Xiaojun , Yang Jerry
Published: July 2002

A catalyst has been synthesized comprising a manganese porphyrin carrying four beta-cyclodextrin groups. It catalyzes the hydroxylation of substrates of appropriate size carrying tert-butylphenyl groups that can hydrophobically bind into the cyclodextrin cavities. In one example as many as 650 catalytic turnovers are seen before the catalyst is oxidatively destroyed, and with a rate comparable to that of typical cytochrome P450 enzymes. In another example, a steroid derivative is regio- and stereoselectively hydroxylated at a single unactivated carbon atom, but more slowly and with fewer turnovers. The carbon attacked is not the most chemically reactive, and the selectivity is determined by the geometry of the catalyst-substrate complex. Nonbinding substrates are not reactive under the conditions used, and substrates with more flexible binding geometries give more than a single product.
Site-specific mutagenesis reveals differences in the structural bases for tight binding of RNase inhibitor to angiogenin and RNase A
Authors: Chen Chang-Zheng , Shapiro Robert
Published: July 2002

RNase inhibitor (RI) binds with extraordinary affinity (
K
i
≈ 10
−13
–10
−16
M) to diverse proteins in the pancreatic RNase superfamily. In the present study, the structural basis for the recognition of two RI ligands, human angiogenin (Ang) and bovine RNase A, has been investigated by site-specific mutagenesis of human RI and Ang. The RI residues examined were those that appear to form strong contacts with RNase A in the crystal structure of the porcine RI·RNase A complex [Kobe, B. & Deisenhofer, J. (1995)
Nature (London)
374, 183–186] that would not be replicated in the Ang complex. Ala substitutions of five of these residues (Glu-287, Lys-320, Glu-401, Cys-408, and Arg-457) were found to have little or no effect on binding of RNase A. In contrast, replacements of Tyr-434, Asp-435, and Tyr-437 and deletion of the C-terminal residue Ser-460 substantially weakened affinity for RNase A: the losses of binding energy associated with the mutations were 5.9, 3.6, 2.6, and 3.5 kcal/mol, respectively. Thus these four residues, which are neighbors in the tertiary structure, appear to constitute a “hot spot” for the RNase A interaction. However, only one of them, Asp-435, was equally important for binding of Ang; the
K
i
increases produced by mutations of the others were 20- to 93-fold smaller for Ang than for RNase A. Consequently, Tyr-434 plays a significant but lesser role in the Ang complex, whereas Tyr-437 and Ser-460 make only minor contributions. Ala mutations of four Ang residues (His-8, Gln-12, Asn-68, and Glu-108) that correspond to RI contacts on RNase A produced no major changes in affinity for RI. These findings indicate that RI uses largely different interactions to achieve its extremely tight binding of RNase A and Ang.
Transport and fate of trifluoroacetate in upland forest and wetland ecosystems
Authors: Likens G. E. , Tartowski S. L. , Berger T. W. , Richey D. G. , Driscoll C. T. , Frank H. G. , Klein A.
Published: July 2002

Although trifluoroacetate (TFA), a breakdown product of chlorofluorocarbon replacements, is being dispersed widely within the biosphere, its ecological fate is largely unknown. TFA was added experimentally to an upland, northern hardwood forest and to a small forest wetland ecosystem within the Hubbard Brook Experimental Forest in New Hampshire. Inputs of TFA were not transported conservatively through these ecosystems; instead, significant amounts of TFA were retained within the vegetation and soil compartments. More TFA was retained by the wetland ecosystem than by the upland forest ecosystem. Using simulation modeling, TFA concentrations were predicted for soil and drainage water until the year 2040.
Transgenic mice expressing human Bcl-2 in their neurons are resistant to 6-hydroxydopamine and 1-methyl-4-phenyl-1,2,3,6- tetrahydropyridine neurotoxicity
Authors: Offen Daniel , Beart Philip M. , Cheung Nam S. , Pascoe Catherine J. , Hochman Ayala , Gorodin Svetlana , Melamed Eldad , Bernard Rozenn , Bernard Ora
Published: July 2002

The protooncogene
bcl-2
inhibits neuronal apoptosis during normal brain development as well as that induced by cytotoxic drugs or growth factor deprivation. We have previously demonstrated that neurons of mice deficient in Bcl-2 are more susceptible to neurotoxins and that the dopamine (DA) level in the striatum after systemic 1-methyl-4-phenyl-1,2,3,6 tetrahydropyridine (MPTP) administration was significantly lower than in wild-type mice. In the present study we have used transgenic mice overexpressing human Bcl-2 under the control of neuron-specific enolase promoter (NSE-
hbcl
-2) to test the effects of the neurotoxins 6-hydroxydopamine (6-OHDA) and MPTP on neuronal survival in these mice. Primary cultures of neocortical neurons from normal and transgenic mice were exposed to these dopaminergic neurotoxins. Addition of 6-OHDA resulted in cell death of essentially all neurons from normal mice. In contrast, in cultures generated from heterozygous NSE-
hbcl-2
transgenic mice, only 69% of the cells died while those generated from homozygous transgenic mice were highly resistant and exhibited only 34% cell death. A similar effect was observed with neurons treated with MPP
+
. Moreover, while the striatal dopamine level after MPTP injections was reduced by 32% in the wild type, the concentration remained unchanged in the NSE-
hbcl-2
heterozygous mice. In contrast levels of glutathione-related enzymes were unchanged. In conclusion, overexpression of Bcl-2 in the neurons provided protection, in a dose-dependent manner, against neurotoxins known to selectively damage dopaminergic neurons. This study provides ideas for inhibition of neuronal cell death in neurodegenerative diseases and for the development of efficient neuroprotective gene therapy.
MEKK1 activates both IκB kinase α and IκB kinase β
Authors: Lee Frank S. , Peters Robert T. , Dang Luan C. , Maniatis Tom
Published: July 2002

A critical step in the signal-induced activation of the transcription factor NF-κB is the site-specific phosphorylation of its inhibitor, IκB, that targets the latter for degradation by the ubiquitin–proteasome pathway. We have previously shown that mitogen-activated protein kinase/ERK kinase kinase 1 (MEKK1) can induce both this site-specific phosphorylation of IκBα at Ser-32 and Ser-36
in vivo
and the activity of a high molecular weight IκB kinase complex
in vitro
. Subsequently, others have identified two proteins, IκB kinase α (IKK-α) and IκB kinase β (IKK-β), that are present in a tumor necrosis factor α-inducible, high molecular weight IκB kinase complex. These kinases are believed to directly phosphorylate IκB based on the examination of the kinase activities of IKK immunoprecipitates, but more rigorous proof of this has yet to be demonstrated. We show herein that recombinant IKK-α and IKK-β can, in fact, directly phosphorylate IκBα at Ser-32 and Ser-36, as well as homologous residues in IκBβ
in vitro
, and thus are bona fide IκB kinases. We also show that MEKK1 can induce the activation of both IKK-α and IKK-β
in vivo
. Finally, we show that IKK-α is present in the MEKK1-inducible, high molecular weight IκB kinase complex and treatment of this complex with MEKK1 induces phosphorylation of IKK-α
in vitro
. We conclude that IKK-α and IKK-β can mediate the NF-κB-inducing activity of MEKK1.
Moieties in an RNA promoter specifically recognized by a viral RNA-dependent RNA polymerase
Authors: Siegel Robert W. , Bellon Laurent , Beigelman Leonid , Kao C. Cheng
Published: July 2002

RNAs 33 nucleotides in length can direct accurate initiation of subgenomic RNA synthesis by the brome mosaic virus RNA-dependent RNA polymerase (RdRp), provided that the native sequences are maintained at five positions: −17, −14, −13, −11, and the +1 initiation site. The functional groups in the bases of these essential nucleotides required to interact with RdRp were examined by using chemically synthesized RNAs containing base analogs at each of the five positions. Analysis using a template competition assay revealed that the mode of recognition for the initiation nucleotide (+1) is distinct from that of the other essential nucleotides in the promoter. Competition experiments also determined that three template nucleotides are sufficient for stable interaction with RdRp. These results identify base moieties in the brome mosaic virus subgenomic promoter required for efficient RNA synthesis and support the hypothesis that the recognition of a RNA promoter by a viral RdRp is analogous to the recognition of DNA promoters by DNA-dependent RNA polymerases.
T helper type 2 inflammatory disease in the absence of interleukin 4 and transcription factor STAT6
Authors: Dent Alexander L. , Hu-Li Jane , Paul William E. , Staudt Louis M.
Published: July 2002

An important signaling pathway for the differentiation of T helper type 2 (TH2) cells from uncommitted CD4 T cell precursors is activation of the STAT6 transcription factor by interleukin 4 (IL-4). The protooncogene BCL-6 is also involved in TH2 differentiation, as BCL-6 −/− mice develop an inflammation of the heart and lungs associated with an overproduction of TH2 cells. Surprisingly, IL-4 −/− BCL-6 −/− and STAT6 −/− BCL-6 −/− double-mutant mice developed the same TH2-type inflammation of the heart and lungs as is characteristic of BCL-6 −/− mice. Furthermore, a TH2 cytokine response developed in STAT6 −/− BCL-6 −/− and IL-4 −/− BCL-6 −/− mice after immunization with a conventional antigen in adjuvant. In contrast to thesein vivofindings, STAT6 was required for thein vitrodifferentiation of BCL-6 −/− T cells into TH2 cells. BCL-6, a transcriptional repressor that can bind to the same DNA binding motifs as STAT transcription factors, seems to regulate TH2 responsesin vivoby a pathway independent of IL-4 and STAT6.
Chromosomal drive and the evolution of meiotic nondisjunction and trisomy in humans
Authors: Day Troy , Taylor Peter D.
Published: July 2002

Trisomy is a genetic abnormality of considerable medical importance. The most familiar example is trisomy 21, which causes Down Syndrome [Cummings, M. R. (1988)
Human Heredity: Principles and Issues
(West Publishing Company, New York)]. In a classic paper, Axelrod and Hamilton [Axelrod, R. & Hamilton, W. D. (1981)
Science
211, 1390–1396] offered a chromosomal drive (CD) hypothesis based on the game iterated prisoner’s dilemma (IPD) to explain the evolution of an increased frequency of trisomic pregnancies with maternal age. In this paper we explore this hypothesis and its predictions in detail. On closer examination we find that IPD does not provide an adequate model for the CD hypothesis. Therefore, we develop a more suitable model and explore the conditions necessary for it to explain maternal age-dependent trisomy. Our results demonstrate that a relationship between the decay of a female’s reproductive potential and chromosomal drive must exist for the CD hypothesis to work. With appropriate parameter values, a comparison of model predictions with empirical estimates for the age-dependence of trisomy reveals a striking correspondence. We point out a close correspondence between other predictions made by the CD hypothesis and empirical observations, as well.
The Ig mutator is dependent on the presence, position, and orientation of the large intron enhancer
Authors: Bachl Juergen , Olsson Carina , Chitkara Nishay , Wabl Matthias
Published: July 2002

Hypermutation at the Ig loci is confined to the area between the promoter and the intronic enhancer, which includes the rearranged variable region gene segment. We identified factors that contribute to the site-specificity at the heavy chain locus. We found that distance from both the promoter and the intronic enhancer is crucial in hypermutation. The presence of the enhancer is required, and, in contrast to its definition for transcriptional activity, its effect is orientation-sensitive.
An additional role for the F-box motif: Gene regulation within the
Neurospora crassa
sulfur control network
Authors: Kumar Anuj , Paietta John V.
Published: July 2002

The F-box represents a protein motif originally identified as a conserved amino-terminal domain within the
Neurospora crassa
negative regulator
sulfur controller-2
. Recently, F-boxes have been found within a number of cell cycle regulatory proteins, where they mediate ubiquitin-driven proteolytic events required for major cell cycle transitions. F-box function, however, is not restricted solely to cell cycle pathways. Here we present evidence expanding F-box function to encompass gene regulatory processes independent of the cell cycle through
in vivo
analysis of an F-box acting within the
N. crassa
sulfur regulatory network. The
Neurospora
sulfur circuit features a set of regulatory genes acting to modulate gene expression based on environmental sulfur conditions. These sulfur regulatory genes include
cys-3
+
, which encodes a basic region-leucine zipper transcriptional activator, as well as the negative regulatory gene
scon-2
+
. Through site-directed mutagenesis of the SCON2 F-box, we have generated a sulfur auxotrophic phenotype previously unobserved in any
scon-2
mutant. Using Northern analysis, we have traced this auxotrophy to a complete shutdown of
cys-3
+
gene expression. We have further analyzed F-box function by constructing a series of chimeric SCON2 proteins containing swapped F-box domains from the yeast transcriptional inhibitor Met30p and the
Candida albicans
cell cycle regulator Cdc4p. The ability of these chimeric proteins to restore partial wild-type sulfur regulation
in vivo
emphasizes the universal nature of this motif and confirms the functional importance of the F-box within noncell cycle regulatory pathways.
Gene therapy with a single chain interleukin 12 fusion protein induces T cell-dependent protective immunity in a syngeneic model of murine neuroblastoma
Authors: Lode Holger N. , Dreier Torsten , Xiang Rong , Varki Nissi M. , Kang Angray S. , Reisfeld Ralph A.
Published: July 2002

A major goal of tumor immunotherapy is the effective eradication of established metastases associated with the induction of a T cell-mediated protective immunity. We achieved this in a poorly immunogenic murine neuroblastoma model by gene therapy with a single chain interleukin 12 (scIL-12) fusion protein that assures equal expression of its p35 and p40 subunits. Thus, NXS2 hybrid neuroblastoma cells (C1300 × dorsal root ganglion cells), which form experimental bone marrow and liver metastases in syngeneic A/J mice, were transduced with a gene encoding murine interleukin 12, monomerized by introduction of a protein linker between the p35 and p40 protein chains of this heterodimeric cytokine. We demonstrate for the first time that subcutaneous vaccination with these transduced cells induces a protective immunity, as indicated by the complete absence of liver and bone marrow metastasis after challenge with NXS2 wild-type tumor cells. Furthermore, vaccination of animals with established liver and bone marrow metastases completely eradicated liver metastases and suppressed bone marrow metastases. The local and systemic immune response against scIL-12-transduced NXS2 cells is largely dependent on CD8
+
T cells. This was demonstrated
in vivo
by depletion of immunocompetent A/J mice with monoclonal anti-CD4 and anti-CD8 antibodies and
in vitro
by specific major histocompatibility complex, class I-restricted CD8
+
T cell-mediated killing of NXS2 and their parental C1300 neuroblastoma cells. In conclusion, we demonstrate successful anti-tumor immunotherapy with an scIL-12 fusion protein that could facilitate clinical application of interleukin 12 gene therapy.
Factors affecting counteraction by methylamines of urea effects on aldose reductase
Authors: Burg Maurice B. , Peters Eugenia M. , Bohren Kurt M. , Gabbay Kenneth H.
Published: July 2002

The concentration of urea in renal medullary cells is high enough to affect enzymes seriously by reducing
V
max
or raising
K
m
, yet the cells survive and function. The usual explanation is that the methylamines found in the renal medulla, namely glycerophosphocholine and betaine, have actions opposite to those of urea and thus counteract its effects. However, urea and methylamines have the similar (not counteracting) effects of reducing both the
K
m
and
V
max
of aldose reductase (EC
1.1.1.21
), an enzyme whose function is important in renal medullas. Therefore, we examined factors that might determine whether counteraction occurs, namely different combinations of assay conditions (pH and salt concentration), methylamines (glycerophosphocholine, betaine, and trimethylamine
N-
oxide), substrates (
dl
-glyceraldehyde and
d
-xylose), and a mutation in recombinant aldose reductase protein (C298A). We find that
V
max
of both wild-type and C298A mutant generally is reduced by urea and/or the methylamines. However, the effects on
K
m
are much more complex, varying widely with the combination of conditions. At one extreme, we find a reduction of
K
m
of wild-type enzyme by urea and/or methylamines that is partially additive, whereas at the other extreme we find that urea raises
K
m
for
d
-xylose of the C298A mutant, betaine lowers the
K
m
, and the two counteract in a classical fashion so that at a 2:1 molar ratio of betaine to urea there is no net effect. We conclude that counteraction of urea effects on enzymes by methylamines can depend on ion concentration, pH, the specific methylamine and substrate, and identity of even a single amino acid in the enzyme.
The human
LARGE
gene from 22q12.3-q13.1 is a new, distinct member of the glycosyltransferase gene family
Authors: Peyrard Myriam , Seroussi Eyal , Sandberg-Nordqvist Ann-Christin , Xie Ya-Gang , Han Fei-Yu , Fransson Ingegerd , Collins John , Dunham Ian , Kost-Alimova Maria , Imreh Stephan , Dumanski Jan P.
Published: July 2002

Meningioma, a tumor of the meninges covering the central nervous system, shows frequent loss of material from human chromosome 22. Homozygous and heterozygous deletions in meningiomas defined a candidate region of >1 Mbp in 22q12.3-q13.1 and directed us to gene cloning in this segment. We characterized a new member of the N-acetylglucosaminyltransferase gene family, the
LARGE
gene. It occupies >664 kilobases and is one of the largest human genes. The predicted 756-aa N-acetylglucosaminyltransferase encoded by
LARGE
displays features that are absent in other glycosyltransferases. The human like-acetylglucosaminyltransferase polypeptide is much longer and contains putative coiled-coil domains. We characterized the mouse
LARGE
ortholog, which encodes a protein 97.75% identical with the human counterpart. Both genes reveal ubiquitous expression as assessed by Northern blot analysis and
in situ
histochemistry. Chromosomal mapping of the mouse gene reveals that mouse chromosome 8C1 corresponds to human 22q12.3-q13.1. Abnormal glycosylation of proteins and glycosphingolipids has been shown as a mechanism behind an increased potential of tumor formation and/or progression. Human tumors overexpress ganglioside GD3 (NeuAcα2,8NeuAcα2,3Galβ1,4Glc-Cer), which in meningiomas correlates with deletions on chromosome 22. It is the first time that a glycosyltransferase gene is involved in tumor-specific genomic rearrangements. An abnormal function of the human like-acetylglucosaminyltransferase protein may be linked to the development/progression of meningioma by altering the composition of gangliosides and/or by effect(s) on other glycosylated molecules in tumor cells.
Farnesol is utilized for isoprenoid biosynthesis in plant cells via farnesyl pyrophosphate formed by successive monophosphorylation reactions
Authors: Thai Long , Rush Jeffrey S. , Maul Jude E. , Devarenne Timothy , Rodgers Dana L. , Chappell Joseph , Waechter Charles J.
Published: July 2002

The ability of
Nicotiana tabacum
cell cultures to utilize farnesol (F-OH) for sterol and sesquiterpene biosynthesis was investigated. [
3
H]F-OH was readily incorporated into sterols by rapidly growing cell cultures. However, the incorporation rate into sterols was reduced by greater than 70% in elicitor-treated cell cultures whereas a substantial proportion of the radioactivity was redirected into capsidiol, an extracellular sesquiterpene phytoalexin. The incorporation of [
3
H]F-OH into sterols was inhibited by squalestatin 1, suggesting that [
3
H]F-OH was incorporated via farnesyl pyrophosphate (F-P-P). Consistent with this possibility,
N. tabacum
proteins were metabolically labeled with [
3
H]F-OH or [
3
H]geranylgeraniol ([
3
H]GG-OH). Kinase activities converting F-OH to farnesyl monophosphate (F-P) and, subsequently, F-P-P were demonstrated directly by
in vitro
enzymatic studies. [
3
H]F-P and [
3
H]F-P-P were synthesized when exogenous [
3
H]F-OH was incubated with microsomal fractions and CTP. The kinetics of formation suggested a precursor–product relationship between [
3
H]F-P and [
3
H]F-P-P. In agreement with this kinetic pattern of labeling, [
32
P]F-P and [
32
P]F-P-P were synthesized when microsomal fractions were incubated with F-OH and F-P, respectively, with [γ-
32
P]CTP serving as the phosphoryl donor. Under similar conditions, the microsomal fractions catalyzed the enzymatic conversion of [
3
H]GG-OH to [
3
H]geranylgeranyl monophosphate and [
3
H]geranylgeranyl pyrophosphate ([
3
H]GG-P-P) in CTP-dependent reactions. A novel biosynthetic mechanism involving two successive monophosphorylation reactions was supported by the observation that [
3
H]CTP was formed when microsomes were incubated with [
3
H]CDP and either F-P-P or GG-P-P, but not F-P. These results document the presence of at least two CTP-mediated kinases that provide a mechanism for the utilization of F-OH and GG-OH for the biosynthesis of isoprenoid lipids and protein isoprenylation.
Intranuclear delivery of an antiviral peptide mediated by the B subunit of
Escherichia coli
heat-labile enterotoxin
Authors: Loregian Arianna , Papini Emanuele , Satin Barbara , Marsden Howard S. , Hirst Timothy R. , Palù Giorgio
Published: July 2002

We report an intracellular peptide delivery system capable of targeting specific cellular compartments. In the model system we constructed a chimeric protein consisting of the nontoxic B subunit of
Escherichia coli
heat-labile enterotoxin (EtxB) fused to a 27-mer peptide derived from the DNA polymerase of herpes simplex virus 1. Viral DNA synthesis takes places in the nucleus and requires the interaction with an accessory factor, UL42, encoded by the virus. The peptide, designated Pol, is able to dissociate this interaction. The chimeric protein, EtxB-Pol, retained the functional properties of both EtxB and peptide components and was shown to inhibit viral DNA polymerase activity
in vitro
via disruption of the polymerase-UL42 complex. When added to virally infected cells, EtxB-Pol had no effect on adenovirus replication but specifically interfered with herpes simplex virus 1 replication. Further studies showed that the antiviral peptide localized in the nucleus, whereas the EtxB component remained associated with vesicular compartments. The results indicate that the chimeric protein entered through endosomal acidic compartments and that the Pol peptide was cleaved from the chimeric protein before being translocated into the nucleus. The system we describe is suitable for delivery of peptides that specifically disrupt protein–protein interactions and may be developed to target specific cellular compartments.
Effective dimensional reduction algorithm for eigenvalue problems for thin elastic structures: A paradigm in three dimensions
Authors: Ovtchinnikov Evgueni E. , Xanthis Leonidas S.
Published: July 2002

We present a methodology for the efficient numerical solution of eigenvalue problems of full three-dimensional elasticity for thin elastic structures, such as shells, plates and rods of arbitrary geometry, discretized by the finite element method. Such problems are solved by iterative methods, which, however, are known to suffer from slow convergence or even convergence failure, when the thickness is small. In this paper we show an effective way of resolving this difficulty by invoking a special preconditioning technique associated with the effective dimensional reduction algorithm (EDRA). As an example, we present an algorithm for computing the minimal eigenvalue of a thin elastic plate and we show both theoretically and numerically that it is robust with respect to both the thickness and discretization parameters, i.e. the convergence does not deteriorate with diminishing thickness or mesh refinement. This robustness is sine qua non for the efficient computation of large-scale eigenvalue problems for thin elastic structures.
Cultivating Sympathy: Sophie Condorcet's Letters on Sympathy
Authors: Forget Evelyn L.
Published: July 2002

In 1798, Sophie de Grouchy, the marquise de Condorcet, published a translation of the seventh edition of Adam Smith'sTheory of Moral Sentiments(1792), along with a series of eight “letters” on the subject of sympathy. These letters are, in fact, substantial essays that allow us to discern how she read Smith. Intellectual historians have a tendency to privilege an author's intent, and to read theTheory of Moral Sentimentsin order to determine what Smith actually meant, and how meaning was constructed in the context of a particular intellectual environment. As long ago as 1978, literary theorists such as Wolfgang Iser suggested that a reader's response is at least as interesting a question as an author's intent (Iser 1978). And Sophie de Grouchy is no ordinary reader. Her translation of, and commentary on, Smith's work allow us to see how a theory constructed in the intellectual context of the Scottish Enlightenment would be received by a different intellectual community. While de Grouchy shared much of the background that informed Smith's work, she could not write a commentary on sympathy during the Terror without taking into account recent French political experience and debate. And, I argue, her reading was not merely idiosyncratic, but rather representative of a particular group of intellectuals seized with the problem of adapting Enlightenment theory to the political reality of the Republic.
Abstinent chronic crack‐cocaine and crackcocaine/alcohol abusers evidence normal hippocampal volumes on MRI despite persistent cognitive impairments
Authors: DI SCLAFANI VICTORIA , TRURAN DIANA L. , BLOOMER COURTNAY , TOLOU‐SHAMS MARINA , CLARK H. WESTLEY , NORMAN DAVID , HANNAUER DAVID , FEIN GEORGE
Published: July 2002

We measured hippocampal volumes and cognitive functioning in crack‐cocaine and crack‐cocaine/alcoholdependent subjects (abstinent approximately 10–12 weeks) compared to age‐matched controls. Cognitive function was evaluated using the computerized MicroCog Assessment of Cognitive Functioning (which includes tests of explicit, declarative memory subserved by the hippocampus). The hippocampal volumes were quantified on T1‐weighted MRIs and were expressed as a proportion of intracranial vault volume. Both subjects and controls showed the larger right versus left hippocampal volume expected in normal anatomy, but we found no differences in hippocampal volume between any of the groups. However, both abstinent cocaine‐dependent subjects and abstinent cocaine/alcohol‐dependent subjects showed persistent cognitive impairments, including deficits in explicit memory. Our results suggest that either: (1) the hippocampus is resistant to structural volume loss in young and middle‐aged cocaine or cocaine/alcohol‐dependent subjects, (2) the hippocampal volume loss suffered by young and middle‐aged cocaine or cocaine/alcohol‐dependent subjects resolves after approximately 3 months of abstinence, or (3) hippocampal atrophy is obscured by the process of gliosis. Further, the cognitive impairments persisting in these abstinent cocaine and cocaine/alcohol‐dependent samples may (1) be unrelated to hippocampal function or (2) be associated with abnormal hippocampal function that is not reflected in MRI measures of overall hippocampal atrophy.
Two C‐Terminal Variants of NBC4, a New Member of the Sodium Bicarbonate Cotransporter Family: Cloning, Characterization, and Localization
Authors: Pushkin Alexander , Abuladze Natalia , Newman Debra , Lee Ivan , Xu Gouping , Kurtz Ira
Published: July 2002

We report the cloning, characterization, and chromosomal assignment of a new member of the sodium bicarbonate cotransporter (NBC) family, NBC4. The NBC4 gene was mapped to chromosome 2p13 and is a new candidate gene for Alstrom syndrome. Two variants of the transporter have been isolated from human testis and heart, which differ in their C termini. NBC4a encodes a 1137‐residue polypeptide and is widely expressed in various tissues, including liver, testis, and spleen. NBC4b is identical to NBC4a except that it has a 16‐nucleotide insert, creating a C‐terminal frame shift. NBC4b encodes a 1074‐residue polypeptide and is highly expressed in heart. Amino acids 1‐1046 are common to both NBC4 variants. NBC4a has two protein‐interacting domains that are lacking in NBC4b: a proline‐rich sequence, PPPSVIKIP (amino acids 1102‐1110), and a consensus PDZ‐interacting domain, SYSL (1134‐1137). NBC4b lacks the stretch of charged residues present in the C terminus of NBC4a and other members of the NBC family.Unlike other members of the NBC family, both NBC4a and NBC4b have a unique glycine‐rich region (amino acids 440‐ 469). In comparison with other members of the bicarbonate transport superfamily, NBC4a and NBC4b are most similar structurally to the electrogenic sodium bicarbonate cotransporters (NBC1).