bluuebunny/crossref_metadata_2025 huggingface dataset:
Discontinuous epitopes of hepatitis B surface antigen derived from a filamentous phage peptide library.
Authors: Chen Y C , Delbrook K , Dealwis C , Mimms L , Mushahwar I K , Mandecki W
Published: July 2002

The structure of the small hepatitis B virus surface antigen (HBsAg) was investigated by epitope mapping of four anti-HBsAg monoclonal antibodies (mAbs). Amino acid sequences of epitopes were derived from affinity-enrichment experiments (biopanning) using a filamentous phage peptide library. The library consists of 10(9) different clones bearing a 30-residue peptide fused to gene III. Sequence homologies between peptides obtained from panning the library against the antibodies and the native HBsAg sequence allowed for precise description of the binding regions. Three of four mAbs were found to bind to distinct discontinuous epitopes between amino acid residues 101 and 207 of HBsAg. The fourth mAb was demonstrated to bind to residues 121-124. The sequence data are supported by ELISA assays demonstrating the binding of the HBsAg-specific peptides on filamentous phage to mAbs. The sequence data were used to map the surface of HBsAg and to derive a topological model for the alpha-carbon trace of the 101-207 region of HBsAg. The approach should be useful for other proteins for which the crystal structure is not available but a representative set of mAbs can be obtained.
Chaperonin-facilitated protein folding: optimization of rate and yield by an iterative annealing mechanism.
Authors: Todd M J , Lorimer G H , Thirumalai D
Published: July 2002

We develop a heuristic model for chaperonin-facilitated protein folding, the iterative annealing mechanism, based on theoretical descriptions of "rugged" conformational free energy landscapes for protein folding, and on experimental evidence that (i) folding proceeds by a nucleation mechanism whereby correct and incorrect nucleation lead to fast and slow folding kinetics, respectively, and (ii) chaperonins optimize the rate and yield of protein folding by an active ATP-dependent process. The chaperonins GroEL and GroES catalyze the folding of ribulose bisphosphate carboxylase at a rate proportional to the GroEL concentration. Kinetically trapped folding-incompetent conformers of ribulose bisphosphate carboxylase are converted to the native state in a reaction involving multiple rounds of quantized ATP hydrolysis by GroEL. We propose that chaperonins optimize protein folding by an iterative annealing mechanism; they repeatedly bind kinetically trapped conformers, randomly disrupt their structure, and release them in less folded states, allowing substrate proteins multiple opportunities to find pathways leading to the most thermodynamically stable state. By this mechanism, chaperonins greatly expand the range of environmental conditions in which folding to the native state is possible. We suggest that the development of this device for optimizing protein folding was an early and significant evolutionary event.
Gene repression by the ferric uptake regulator in Pseudomonas aeruginosa: cycle selection of iron-regulated genes.
Authors: Ochsner U A , Vasil M L
Published: July 2002

The expression of at least 24 distinct genes of Pseudomonas aeruginosa PAO1 is under direct control of the "ferric uptake regulator" (Fur). Novel targets of the Fur protein were isolated in a powerful SELEX (systematic evolution of ligands by exponential enrichment)-like cycle selection consisting of in vitro DNA-Fur interaction, binding to anti-Fur antibody, purification on protein G, and PCR amplification. DNA fragments obtained after at least three exponential enrichment cycles were cloned and subjected to DNA mobility-shift assays and DNase I footprint analyses to verify the specific interaction with the Fur protein in vitro. Iron-dependent expression of the corresponding genes in vivo was monitored by RNase protection analysis. In total, 20 different DNA fragments were identified which represent actual Pseudomonas iron-regulated genes (PIGs). While four PIGs are identical to already known genes (pfeR, pvdS, tonB, and fumC, respectively), 16 PIGs represent previously unknown genes. Homology studies of the putative proteins encoded by the PIGs allowed us to speculate about their possible function. Two PIG products were highly similar to siderophore receptors from various species, and three PIG products were significantly homologous to alternative sigma factors. Furthermore, homologs of the Escherichia coli ORF1-tolQ, nuoA, stringent starvation protein Ssp, and of a two-component regulatory system similar to the Pseudomonas syringae LemA sensor kinase were identified. The putative gene products of seven additional PIGs did not show significant homologies to any known proteins. The PIGs were mapped on the P.aeruginosa chromosome. Their possible role in iron metabolism and virulence of P. aeruginosa is discussed.
A novel pair of immunoglobulin-like receptors expressed by B cells and myeloid cells
Authors: Kubagawa Hiromi , Burrows Peter D. , Cooper Max D.
Published: July 2002

An Fcα receptor probe of human origin was used to identify novel members of the Ig gene superfamily in mice.PairedIg-likereceptors, named PIR-A and PIR-B, are predicted from sequence analysis of the cDNAs isolated from a mouse splenic library. Both type I transmembrane proteins possess similar ectodomains with six Ig-like loops, but have different transmembrane and cytoplasmic regions. The predicted PIR-A protein has a short cytoplasmic tail and a charged Arg residue in the transmembrane region that, by analogy with the FcαR relative, suggests the potential for association with an additional transmembrane protein to form a signal transducing unit. In contrast, the PIR-B protein has an uncharged transmembrane region and a long cytoplasmic tail containing four potential immunoreceptor tyrosine-based inhibitory motifs. These features are shared by the related killer inhibitory receptors. PIR-A proteins appear to be highly variable, in that predicted peptide sequences differ for seven randomly selected PIR-A clones, whereas PIR-B cDNA clones are invariant. Southern blot analysis with PIR-B and PIR-A-specific probes suggests only onePIR-Bgene and multiplePIR-Agenes. ThePIR-AandPIR-Bgenes are expressed in B lymphocytes and myeloid lineage cells, wherein both are expressed simultaneously. The characteristics of the highly-conservedPIR-AandPIR-Bgenes and their coordinate cellular expression suggest a potential regulatory role in humoral, inflammatory, and allergic responses.
Expansion of a CUG trinucleotide repeat in the 3′ untranslated region of myotonic dystrophy protein kinase transcripts results in nuclear retention of transcripts
Authors: Davis Brigid M. , McCurrach Mila E. , Taneja Krishan L. , Singer Robert H. , Housman David E.
Published: July 2002

Expansion of a CTG trinucleotide repeat in the 3′ untranslated region (UTR) of
DMPK
, the gene encoding myotonic dystrophy protein kinase, induces the dominantly inherited neuromuscular disorder myotonic dystrophy (DM). Transcripts containing the expanded trinucleotide are abundant in differentiated cultured myoblasts, and they are spliced and polyadenylylated normally. However, mutant transcripts never reach the cytoplasm in these nonmitotic cells; instead, they form stable clusters that are tightly linked to the nuclear matrix, which can prevent effective biochemical purification of these transcripts. In DM patients, reduced DMPK protein levels, consequent to nuclear retention of mutant transcripts, are probably a cause of disease development. Formation of nuclear foci is a novel mechanism for preventing transcript export and effecting a loss of gene function.
DNA variation at the
Sod
locus of
Drosophila melanogaster
: An unfolding story of natural selection
Authors: Hudson Richard R. , Sáez Alberto G. , Ayala Francisco J.
Published: July 2002

Patterns of variation at the
Sod
locus of
Drosophila melanogaster
suggest that the protein
polymorphism at this locus has very recently arisen. In addition, it
appears that a previously rare DNA variant has been recently and
rapidly driven to intermediate frequency. From the size of the region
(>20 kb) that has been swept along with this rare variant, and
patterns of linkage disequilibrium in the region, it is inferred that
strength of selection was large (s > 0.01) and that the sweep
occurred more than 25,000 generations ago. In addition, there are
striking similarities to patterns of variation observed at the
Est6
and
Est-P
loci, which are located
approximately 1,000 kb from
Sod
.
Fusion genes resulting from alternative chromosomal translocations are overexpressed by gene-specific mechanisms in alveolar rhabdomyosarcoma
Authors: Davis Richard J. , Barr Frederic G.
Published: July 2002

Chromosomal translocations identified in hematopoietic and solid tumors result in deregulated expression of protooncogenes or creation of chimeric proteins with tumorigenic potential. In the pediatric solid tumor alveolar rhabdomyosarcoma, a consistent t(2;13)(q35;q14) or variant t(1;13)(p36;q14) translocation generates PAX3-FKHR or PAX7-FKHR fusion proteins, respectively. In this report, we demonstrate that in addition to functional alterations these translocations are associated with fusion product overexpression. Furthermore, PAX3-FKHR and PAX7-FKHR overexpression occurs by distinct mechanisms. Transcription of
PAX3-FKHR
is increased relative to wild-type
PAX3
by a copy number-independent process. In contrast,
PAX7-FKHR
overexpression results from fusion gene amplification. Thus, gene-specific mechanisms were selected to overexpress
PAX3-FKHR
and
PAX7-FKHR
in alveolar rhabdomyosarcoma, presumably due to differences in regulation between the wild-type loci. We postulate that these overexpression mechanisms ensure a critical level of gene product for the oncogenic effects of these fusions.
A Z-DNA binding domain present in the human editing enzyme, double-stranded RNA adenosine deaminase
Authors: Herbert Alan , Alfken Jens , Kim Yang-Gyun , Mian I. Saira , Nishikura Kazuko , Rich Alexander
Published: July 2002

Editing of RNA changes the read-out of information from DNA by altering the nucleotide sequence of a transcript. One type of RNA editing found in all metazoans uses double-stranded RNA (dsRNA) as a substrate and results in the deamination of adenosine to give inosine, which is translated as guanosine. Editing thus allows variant proteins to be produced from a single pre-mRNA. A mechanism by which dsRNA substrates form is through pairing of intronic and exonic sequences before the removal of noncoding sequences by splicing. Here we report that the RNA editing enzyme, human dsRNA adenosine deaminase (DRADA1, or ADAR1) contains a domain (Zα) that binds specifically to the left-handed Z-DNA conformation with high affinity (KD= 4 nM). As formation of Z-DNAin vivooccurs 5′ to, or behind, a moving RNA polymerase during transcription, recognition of Z-DNA by DRADA1 provides a plausible mechanism by which DRADA1 can be targeted to a nascent RNA so that editing occurs before splicing. Analysis of sequences related to Zα has allowed identification of motifs common to this class of nucleic acid binding domain.
Similarities in normal and binocularly rivalrous viewing
Authors: Andrews Timothy J. , Purves Dale
Published: July 2002

We report here a series of observations—most of which the reader can experience directly—showing that distinct components of patterned visual stimuli (orthogonal lines of a different hue) vary in perception as sets. Although less frequent and often less complete, these perceptual fluctuations in normal viewing are otherwise similar to the binocular rivalry experienced when incompatible scenes are presented dichoptically.
Characterization of human telomerase complex
Authors: Ramakrishnan Shyam , Sharma Harsh W. , Farris A. Darise , Kaufman Kenneth M. , Harley John B. , Collins Kathleen , Pruijn Ger J. M. , van Venrooij Walther J. , Martin Mitchell L. , Narayanan Ramaswamy
Published: July 2002

Telomerase, a ribonucleoprotein complex, adds hexameric repeats called “telomeres” to the growing ends of chromosomal DNA. Characterization of mammalian telomerase has been elusive because of its low level of expression. We describe a bioinformatics approach to enrich and characterize the human telomerase complex. Using local sequence homology search methods, we detected similarity of the
Tetrahymena
p80 subunit of telomerase with the autoantigen Ro60. Antibodies to Ro60 immunoprecipitated the telomerase activity. Ro60 and p80 proteins were cross-recognizable by antibodies to either protein. Telomerase activity and the RNA component of telomerase complex were localized to a doublet in a native gel from the Ro60 antibody-precipitated material. The enriched material showed specific binding to a TTA GGG probe
in vitro
in an RNA template-dependent manner. Polyclonal antibodies to the doublet also immunoprecipitated the telomerase activity. These results suggest an evolutionary conservation of the telomerase proteins.
Specific atomic groups and RNA helix geometry in acceptor stem recognition by a tRNA synthetase
Authors: Beuning Penny J. , Yang Fan , Schimmel Paul , Musier-Forsyth Karin
Published: July 2002

Oligonucleotides that recapitulate the acceptor stems of tRNAs are substrates for aminoacylation by many tRNA synthetases
in vitro
, even though these substrates are missing the anticodon trinucleotides of the genetic code. In the case of tRNA
Ala
a single acceptor stem G⋅U base pair at position 3·70 is essential, based on experiments where the wobble pair has been replaced by alternatives such as I⋅U, G⋅C, and A⋅U, among others. These experiments led to the conclusion that the minor-groove free 2-amino group (of guanosine) of the G⋅U wobble pair is essential for charging. Moreover, alanine-inserting tRNAs (amber suppressors) that replace G⋅U with mismatches such as G⋅A and C⋅A are partially active
in vivo
and can support growth of an
Escherichia coli
tRNA
Ala
knockout strain, leading to the hypothesis that a helix irregularity and nucleotide functionalities are important for recognition. Herein we investigate the charging
in vitro
of oligonucleotide and full-length tRNA substrates that contain mismatches at the position of the G⋅U pair. Although most of these substrates have undetectable activity, G⋅A and C⋅A variants retain some activity, which is, nevertheless, reduced by at least 100-fold. Thus, the
in vivo
assays are much less sensitive to large changes in aminoacylation kinetic efficiency of 3·70 variants than is the
in vitro
assay system. Although these functional data do not clarify all of the details, it is now clear that specific atomic groups are substantially more important in determining kinetic efficiency than is a helical distortion. By implication, the activity of mutant tRNAs measured in the
in vivo
assays appears to be more dependent on factors other than aminoacylation kinetic efficiency.
Isolation and bacterial expression of a sesquiterpene synthase cDNA clone from peppermint (
Mentha
x
piperita
, L.) that produces the aphid alarm pheromone (
E
)-β-farnesene
Authors: Crock John , Wildung Mark , Croteau Rodney
Published: July 2002

(
E
)-β-Farnesene is a sesquiterpene semiochemical that is used extensively by both plants and insects for communication. This acyclic olefin is found in the essential oil of peppermint (
Mentha
x
piperita
) and can be synthesized from farnesyl diphosphate by a cell-free extract of peppermint secretory gland cells. A cDNA from peppermint encoding (
E
)-β-farnesene synthase was cloned by random sequencing of an oil gland library and was expressed in
Escherichia coli
. The corresponding synthase has a deduced size of 63.8 kDa and requires a divalent cation for catalysis (
K
m
for Mg
2+
≈ 150 μM;
K
m
for Mn
2+
≈ 7 μM). The sesquiterpenoids produced by the recombinant enzyme, as determined by radio-GC and GC-MS, are (
E
)-β-farnesene (85%), (
Z
)-β-farnesene (8%), and δ-cadinene (5%) with the native C15 substrate farnesyl diphosphate (
K
m
≈ 0.6 μM;
V
rel
= 100) and Mg
2+
as cofactor, and (
E
)-β-farnesene (98%) and (
Z
)-β-farnesene (2%) with Mn
2+
as cofactor (
V
rel
= 80). With the C
10
analog, GDP, as substrate (
K
m
= 1.5 μM;
V
rel
= 3 with Mg
2+
as cofactor), the monoterpenes limonene (48%), terpinolene (15%), and myrcene (15%) are produced.
A hepatic lipase (
LIPC
) allele associated with high plasma concentrations of high density lipoprotein cholesterol
Authors: Guerra Rudy , Wang Jinping , Grundy Scott M. , Cohen Jonathan C.
Published: July 2002

Genetic factors strongly influence interindividual variation in plasma high density lipoprotein cholesterol (HDL-C) levels, but the specific genetic polymorphisms that confer heritable variation in HDL-C levels have not been identified. In this study we examined the relationship between polymorphism in
LIPC
, the gene encoding hepatic lipase, and plasma HDL-C concentrations using a sequential approach comprising linkage analysis, DNA sequencing, and association studies. Linkage studies in 1465 American white subjects from 218 nuclear families indicated that allelic variation at, or closely linked to, the hepatic lipase gene accounts for a significant fraction (≈25%) of the variation in plasma HDL-C concentrations. The hepatic lipase gene was then sequenced in selected individuals, and four novel polymorphisms were identified in the 5′ flanking region of the gene. These polymorphisms were in complete linkage disequilibrium and thus identified a single novel allele. Association studies indicated that heterozygosity for the rare allele was associated with modestly increased concentrations of plasma HDL-C (41 ± 11 vs. 37 ± 10 mg/dl,
P
< 0.05) and apolipoprotein AI in men (131 ± 23 vs. 122 ± 21 mg/dl,
P
< 0.05) but not in women. Homozygosity for the rare allele was associated with markedly higher plasma HDL-C (63 ± 3 mg/dl) and apolipoprotein AI (153 ± 9 mg/dl) concentrations in men. The results of the association study were replicated in a second, independently ascertained sample. Taken together, the results of the linkage and association studies provide strong evidence that genetic variation in hepatic lipase activity is a major determinant of plasma HDL-C levels.
Cloning and characterization of human protease-activated receptor 4
Authors: Xu Wen-feng , Andersen Henrik , Whitmore Theodore E. , Presnell Scott R. , Yee David P. , Ching Andrew , Gilbert Teresa , Davie Earl W. , Foster Donald C.
Published: July 2002

Protease-activated receptors 1–3 (PAR1, PAR2, and PAR3) are members of a unique G protein-coupled receptor family. They are characterized by a tethered peptide ligand at the extracellular amino terminus that is generated by minor proteolysis. A partial cDNA sequence of a fourth member of this family (PAR4) was identified in an expressed sequence tag database, and the full-length cDNA clone has been isolated from a lymphoma Daudi cell cDNA library. The ORF codes for a seven transmembrane domain protein of 385 amino acids with 33% amino acid sequence identity with PAR1, PAR2, and PAR3. A putative protease cleavage site (Arg-47/Gly-48) was identified within the extracellular amino terminus. COS cells transiently transfected with PAR4 resulted in the formation of intracellular inositol triphosphate when treated with either thrombin or trypsin. A PAR4 mutant in which the Arg-47 was replaced with Ala did not respond to thrombin or trypsin. A hexapeptide (GYPGQV) representing the newly exposed tethered ligand from the amino terminus of PAR4 after proteolysis by thrombin activated COS cells transfected with either wild-type or the mutant PAR4. Northern blot showed that PAR4 mRNA was expressed in a number of human tissues, with high levels being present in lung, pancreas, thyroid, testis, and small intestine. By fluorescencein situhybridization, the human PAR4 gene was mapped to chromosome 19p12.
Proteinase-activated receptor 2 (PAR
2
)-activating peptides: Identification of a receptor distinct from PAR
2
that regulates intestinal transport
Authors: Vergnolle Nathalie , Macnaughton Wallace K. , Al-Ani Bahjat , Saifeddine Mahmoud , Wallace John L. , Hollenberg Morley D.
Published: July 2002

The effects of PAR
2
-activating PAR
2
-activating peptides, SLIGRL (SL)-NH
2
, and
trans
-cinnamoyl-LIGRLO (tc)-NH
2
were compared with the action of trypsin, thrombin, and the PAR
1
selective-activating peptide: Ala-parafluoroPhe-Arg-cyclohexylAla-Citrulline-Tyr (Cit)-NH
2
for stimulating intestinal ion transport. These agonists were added to the serosa of stripped rat jejunum segments mounted in Ussing chambers, and short circuit current (Isc) was used to monitor active ion transport. The relative potencies of these agonists also were evaluated in two bioassays specific for the activation of rat PAR
2
: a cloned rat PAR
2
cell calcium-signaling assay (PAR
2
-KNRK cells) and an aorta ring relaxation (AR) assay. In the Isc assay, all agonists, except thrombin, induced an Isc increase. The SL-NH
2
-induced Isc changes were blocked by indomethacin but not by tetrodotoxin. The relative potencies of the agonists in the Isc assay (trypsin≫SL-NH
2
>tc-NH
2
>Cit-NH
2
) were strikingly different from their relative potencies in the cloned PAR
2
-KNRK cell calcium assay (trypsin≫>tc-NH
2
≅ SL-NH
2
≫>Cit-NH
2
) and in the AR assay (trypsin≫>tc-NH
2
≅ SL-NH
2
). Furthermore, all agonists were maximally active in the PAR
2
-KNRK cell and AR assays at concentrations that were one (PAR
2
-activating peptides) or two (trypsin) orders of magnitude lower than those required to activate intestinal transport. Based on the distinct potency profile for these agonists and the considerable differences in the concentration ranges required to induce an Isc effect in the intestinal assay compared with the PAR
2
-KNRK and AR assays, we conclude that a proteinase-activated receptor, pharmacologically distinct from PAR
2
and PAR
1
, is present in rat jejunum and regulates intestinal transport via a prostanoid-mediated mechanism.
Zf9, a Kruppel-like transcription factor up-regulated
in vivo
during early hepatic fibrosis
Authors: Ratziu Vlad , Lalazar Avraham , Wong Linda , Dang Qi , Collins Colin , Shaulian Eitan , Jensen Susan , Friedman Scott L.
Published: July 2002

Wound repair in the liver induces altered gene expression in stellate cells (resident mesenchymal cells) in a process known as “activation.” A zinc finger transcription factor cDNA,
zf9
, was cloned from rat stellate cells activated
in vivo
. Zf9 expression and biosynthesis are increased markedly in activated cells
in vivo
compared with cells from normal rats (“quiescent” cells). The factor is localized to the nucleus and the perinuclear zone in activated but not quiescent cells. Zf9 mRNA also is expressed widely in nonhepatic adult rat tissues and the fetal liver. The
zf9
nucleotide sequence predicts a member of the Kruppel-like family with a unique N-terminal domain rich in serine–proline clusters and leucines. The human
zf9
gene maps to chromosome 10P near the telomere. Zf9 binds specifically to a DNA oligonucleotide containing a GC box motif. The N-terminal domain of Zf9 (amino acids 1–201) is transactivating in the chimeric GAL4 hybrid system. In
Drosophila schneider
cells, full length Zf9 transactivates a reporter construct driven by the SV40 promoter/enhancer, which contains several GC boxes. A physiologic role for Zf9 is suggested by its transactivation of a collagen α1(I) promoter reporter. Transactivation of collagen α1(I) by Zf9 is context-dependent, occurring strongly in stellate cells, modestly in Hep G2 cells, and not at all in
D. schneider
cells. Our results suggest that Zf9 may be an important signal in hepatic stellate cell activation after liver injury.
Determination of transmembrane topology of an inward-rectifying potassium channel from
Arabidopsis thaliana
based on functional expression in
Escherichia coli
Authors: Uozumi Nobuyuki , Nakamura Tatsunosuke , Schroeder Julian I. , Muto Shoshi
Published: July 2002

We report here that the inward-rectifying potassium channels KAT1
and AKT2 were functionally expressed in K
+
uptake-deficient
Escherichia coli
. Immunological assays showed that KAT1
was translocated into the cell membrane of
E. coli
.
Functional assays suggested that KAT1 was inserted topologically
correctly into the cell membrane. In control experiments, the inactive
point mutation in KAT1, T256R, did not complement for K
+
uptake in
E. coli
. The inward-rectifying K
+
channels of plants share a common hydrophobic domain comprising at
least six membrane-spanning segments (S1–S6). The finding that a
K
+
channel can be expressed in bacteria was further
exploited to determine the KAT1 membrane topology by a gene fusion
approach using the bacterial reporter enzymes, alkaline phosphatase,
which is active only in the periplasm, and β-galactosidase. The
enzyme activity from the alkaline phosphatase and β-galactosidase
fusion plasmid showed that the widely predicted S1, S2, S5, and S6
segments were inserted into the membrane. Although the S3 segment in
the alkaline phosphatase fusion protein could not function as an export
signal, the replacement of a negatively charged residue inside S3 with
a neutral amino acid resulted in an increase in alkaline phosphatase
activity, which indicates that the alkaline phosphatase was
translocated into the periplasm. For membrane translocation of S3, the
neutralization of a negatively charged residue in S3 may be required
presumably because of pairing with a positively charged residue of S4.
These results revealed that KAT1 has the common six
transmembrane-spanning membrane topology that has been predicted for
the Shaker superfamily of voltage-dependent K
+
channels.
Furthermore, the functional complementation of a bacterial
K
+
uptake mutant in this study is shown to be an
alternative expression system for plant K
+
channel proteins
and a potent tool for their topological analysis.
Mad2 transiently associates with an APC/p55Cdc complex during mitosis
Authors: Wassmann Katja , Benezra Robert
Published: July 2002

Activation of the mitotic checkpoint pathway in response to mitotic spindle damage in eukaryotic cells delays the exit from mitosis in an attempt to prevent chromosome missegregation. One component of this pathway, hsMad2, has been shown in mammalian cells to physically associate with components of a ubiquitin ligase activity (termed the anaphase promoting complex or APC) when the checkpoint is activated, thereby preventing the degradation of inhibitors of the mitotic exit machinery. In the present report, we demonstrate that the inhibitory association between Mad2 and the APC component Cdc27 also takes place transiently during the early stages of a normal mitosis and is lost before mitotic exit. We also show that Mad2 associates with the APC regulatory protein p55Cdc in mammalian cells as has been reported in yeast. In contrast, however, this complex is present only in nocodazole-arrested or early mitotic cells and is associated with the APC as a Mad2/p55Cdc/Cdc27 ternary complex. Evidence for a Mad2/Cdc27 complex that forms independent of p55Cdc also is presented. These results suggest a model for the regulation of the APC by Mad2 and may explain how the spindle assembly checkpoint apparatus controls the timing of mitosis under normal growth conditions.
Neuroimaging analyses of human working memory
Authors: Smith Edward E. , Jonides John
Published: July 2002

We review a program of research that uses neuroimaging techniques to determine the functional and neural architecture of human working memory. A first set of studies indicates that verbal working memory includes a storage component, which is implemented neurally by areas in the left-hemisphere posterior parietal cortex, and a subvocal rehearsal component, which is implemented by left-hemisphere speech areas, including Broca’s area as well as the premotor and supplementary motor areas. We provide a number of neuroimaging dissociations between the storage and rehearsal areas. A second set of studies focuses on spatial working memory and indicates that it is mediated by a network of predominantly right-hemisphere regions that include areas in posterior parietal, occipital, and frontal cortex. We provide some suggestive evidence that these areas, too, divide into storage and rehearsal regions, with right-hemisphere posterior parietal and premotor regions subserving spatial rehearsal. In a final set of studies, we turn to “executive processes,” metaprocesses that regulate the processing of working-memory contents. We focus on the executive process of inhibition as it is used in verbal working memory. We provide evidence that such inhibition is mediated by the left-hemisphere prefrontal region and that it can be dissociated from verbal storage and rehearsal processes.
The
pir
gene of
Erwinia chrysanthemi
EC16 regulates hyperinduction of pectate lyase virulence genes in response to plant signals
Authors: Nomura Kinya , Nasser William , Kawagishi Hirokazu , Tsuyumu Shinji
Published: July 2002

The plant pathogenic bacterium
Erwinia chrysanthemi
secretes pectate lyase proteins that are important virulence factors attacking the cell walls of plant hosts. Bacterial production of these enzymes is induced by the substrate polypectate-Na (NaPP) and further stimulated by the presence of plant extracts. The bacterial regulator responsible for induction by plant extracts was identified and purified by using a DNA-binding assay with the promoter region of
pelE
that encodes a major pectate lyase. A novel bacterial protein, called Pir, was isolated that produced a specific gel shift of the
pelE
promoter DNA, and the corresponding
pir
gene was cloned and sequenced. The Pir protein contains 272 amino acids with a molecular mass of 30 kDa and appears to function as a dimer. A homology search indicates that Pir belongs to the IclR family of transcriptional regulators. Pir bound to a 35-bp DNA sequence in the promoter region of
pelE
. This site overlaps that of a previously described negative regulator, KdgR. Gel shift experiments showed that the binding of either Pir or KdgR interfered with binding of the other protein.
Sustained secretion of human alpha-1-antitrypsin from murine muscle transduced with adeno-associated virus vectors
Authors: Song Sihong , Morgan Michael , Ellis Tamir , Poirier Amy , Chesnut Kye , Wang Jianming , Brantly Mark , Muzyczka Nicholas , Byrne Barry J. , Atkinson Mark , Flotte Terence R.
Published: July 2002

Recombinant adeno-associated virus (AAV) vectors have been used to transduce murine skeletal muscle as a platform for secretion of therapeutic proteins. The utility of this approach for treating alpha-1-antitrypsin (AAT) deficiency was tested in murine myocytes
in vitro
and
in vivo
. AAV vectors expressing the human AAT gene from either the cytomegalovirus (CMV) promoter (AAV-C-AT) or the human elongation factor 1-α promoter (AAV-E-AT) were examined.
In vitro
in C2C12 murine myoblasts, the expression levels in transient transfections were similar between the two vectors. One month after transduction, however, the human elongation factor 1 promoter mediated 10-fold higher stable human AAT expression than the CMV promoter.
In vivo
transduction was performed by injecting doses of up to 1.4 × 10
13
particles into skeletal muscles of several mouse strains (C57BL/6, BALB/c, and SCID).
In vivo
, the CMV vector mediated higher levels of expression, with sustained serum levels over 800 μg/ml in SCID and over 400 μg/ml in C57BL/6 mice. These serum concentrations are 100,000-fold higher than those previously observed with AAV vectors in muscle and are at levels which would be therapeutic if achieved in humans. High level expression was delayed for several weeks but was sustained for over 15 wk. Immune responses were dependent upon the mouse strain and the vector dosage. These data suggest that recombinant AAV vector transduction of skeletal muscle could provide a means for replacing AAT or other essential serum proteins but that immune responses may be elicited under certain conditions.
Insect juvenile hormone resistance gene homology with the bHLH-PAS family of transcriptional regulators
Authors: Ashok Mudgapalli , Turner Christopher , Wilson Thomas G.
Published: July 2002

Juvenile hormone analog (JHA) insecticides are relatively nontoxic to vertebrates and offer effective control of certain insect pests. Recent reports of resistance in whiteflies and mosquitoes demonstrate the need to identify and understand genes for resistance to this class of insect growth regulators. Mutants of the
Methoprene-tolerant
(
Met
) gene in
Drosophila melanogaster
show resistance to both JHAs and JH, and previous biochemical studies have demonstrated a mechanism of resistance involving an intracellular JH binding-protein that has reduced ligand affinity in
Met
flies. We cloned the
Met
+
gene by transposable
P
-element tagging and found reduced transcript level in several mutant alleles, showing that underproduction of the normal gene product can lead to insecticide resistance. Transformation of
Met
flies with a
Met
+
cDNA resulted in susceptibility to methoprene, indicating that the cDNA encodes a functional
Met
+
protein. MET shows homology to the basic helix-loop-helix (bHLH)-PAS family of transcriptional regulators, implicating MET in the action of JH at the gene level in insects. This family also includes the vertebrate dioxin receptor, a transcriptional regulator known to bind a variety of environmental toxicants. Because JHAs include a diverse array of chemicals with JH activity, a mechanism whereby they can exert effects in insects through a common pathway is suggested.
A cytoplasmic protein, bystin, interacts with trophinin, tastin, and cytokeratin and may be involved in trophinin-mediated cell adhesion between trophoblast and endometrial epithelial cells
Authors: Suzuki Nao , Zara Jane , Sato Takaaki , Ong Edgar , Bakhiet Nouna , Oshima Robert G. , Watson Kellie L. , Fukuda Michiko N.
Published: July 2002

Trophinin and tastin form a cell adhesion molecule complex that potentially mediates an initial attachment of the blastocyst to uterine epithelial cells at the time of implantation. Trophinin and tastin, however, do not directly bind to each other, suggesting the presence of an intermediary protein. The present study identifies a cytoplasmic protein, named bystin, that directly binds trophinin and tastin. Bystin consists of 306 amino acid residues and is predicted to contain tyrosine, serine, and threonine residues in contexts conforming to motifs for phosphorylation by protein kinases. Database searches revealed a 53% identity of the predicted peptide sequence with the
Drosophila bys
(
mrr
) gene. Direct protein–protein interactions of trophinin, tastin, and bystin analyzed by yeast two-hybrid assays and by
in vitro
protein binding assays indicated that binding between bystin and trophinin and between bystin and tastin is enhanced when cytokeratin 8 and 18 are present as the third molecule. Immunocytochemistry of bystin showed that bystin colocalizes with trophinin, tastin, and cytokeratins in a human trophoblastic teratocarcinoma cell, HT-H. It is therefore possible that these molecules form a complex and thus are involved in the process of embryo implantation.
CPF: An orphan nuclear receptor that regulates liver-specific expression of the human cholesterol 7α-hydroxylase gene
Authors: Nitta Masahiro , Ku Sherry , Brown Chaline , Okamoto Arthur Y. , Shan Bei
Published: July 2002

Cholesterol 7α-hydroxylase is the first and rate-limiting enzyme in a pathway through which cholesterol is metabolized to bile acids. The gene encoding cholesterol 7α-hydroxylase,
CYP7A
, is expressed exclusively in the liver. Overexpression of
CYP7A
in hamsters results in a reduction of serum cholesterol levels, suggesting that the enzyme plays a central role in cholesterol homeostasis. Here, we report the identification of a hepatic-specific transcription factor that binds to the promoter of the human
CYP7A
gene. We designate this factor CPF, for
CYP7A
promoter binding factor. Mutation of the CPF binding site within the
CYP7A
promoter abolished hepatic-specific expression of the gene in transient transfection assays. A cDNA encoding CPF was cloned and identified as a human homolog of the
Drosophila
orphan nuclear receptor fushi tarazu F1 (Ftz-F1). Cotransfection of a CPF expression plasmid and a
CYP7A
reporter gene resulted in specific induction of
CYP7A
-directed transcription. These observations suggest that CPF is a key regulator of human
CYP7A
gene expression in the liver.
Identification of the gregarization-associated dark-pigmentotropin in locusts through an albino mutant
Authors: Tawfik Amer I. , Tanaka Seiji , De Loof Arnold , Schoofs Liliane , Baggerman Geert , Waelkens Etienne , Derua Rita , Milner Yoram , Yerushalmi Yoram , Pener M. Paul
Published: July 2002

In response to crowding, locusts develop characteristic black patterns that are well discernible in the gregarious phase at outbreaks. We report here a dark-color-inducing neuropeptide (dark-pigmentotropin) from the corpora cardiaca of two plague locusts,
Schistocerca gregaria
and
Locusta migratoria
. The chromatographic isolation of this neuropeptide was monitored by using a bioassay with an albino mutant of
L. migratoria.
The neurohormone, consisting of 11 amino acids, is identical to [His
7
] corazonin, previously isolated from corpora cardiaca of another acridid without known function. The present results show that even in isolated (solitary) nymphs, [His
7
] corazonin induces gregarious black patterns. Its primary structure shows some similarity with the vertebrate melanophore stimulating hormone.
Hot-spot mutants of p53 core domain evince characteristic local structural changes
Authors: Wong Kam-Bo , DeDecker Brian S. , Freund Stefan M. V. , Proctor Mark R. , Bycroft Mark , Fersht Alan R.
Published: July 2002

Most of the oncogenic mutations in the tumor suppressor p53 map to its DNA-binding (core) domain. It is thus a potential target in cancer therapy for rescue by drugs. To begin to understand how mutation inactivates p53 and hence to provide a structural basis for drug design, we have compared structures of wild-type and mutant p53 core domains in solution by NMR spectroscopy. Structural changes introduced by five hot-spot mutations (V143A, G245S, R248Q, R249S, and R273H) were monitored by chemical-shift changes. Only localized changes are observed for G245S, R248Q, R249S, and R273H, suggesting that the overall tertiary folds of these mutant proteins are similar to that of wild type. Structural changes in R273H are found mainly in the loop–sheet–helix motif and the loop L3 of the core domain. Mutations in L3 (G245S, R248Q, and R249S) introduce structural changes in the loop L2 and L3 as well as terminal residues of strands 4, 9, and 10. It is noteworthy that R248Q, which is often regarded as a contact mutant that affects only interactions with DNA, introduces structural changes as extensive as the other loop L3 mutations (G245S and R249S). These changes suggest that R248Q is also a structural mutant that perturbs the structure of loop L2–L3 regions of the p53 core domain. In contrast to other mutants, replacement of the core residue valine 143 to alanine causes chemical-shift changes in almost all residues in the β-sandwich and the DNA-binding surface. Long-range effects of V143A mutation may affect the specificity of DNA binding.
The yeastHMLI silencer defines a heterochromatin domain boundary by directional establishment of silencing
Authors: Bi Xin , Braunstein Miriam , Shei Gan-Ju , Broach James R.
Published: July 2002

The eukaryotic genome is divided into functional domains defined in part by local differences in chromatin structure and delimited in many cases by boundary elements. TheHMLandHMRloci in the yeastSaccharomyces cerevisiaeare transcriptionally silent chromosome domains. Each locus is bracketed by two cis-acting sequences, designated E and I, that serve to establish and maintain repression of genes within each locus. We show that repression atHMLis uniformly high between E and I but decreases sharply beyond I. The region of repression atHMLgenerally correlates with the domain of histone hypoacetylation. Despite the sharp definition of the boundaries ofHML, no sequence capable of blocking the spread of heterochromatin resides in the sequences flankingHML. We find, though, that inverting the orientation of I increases silencing outside ofHMLwhile weakening silencing withinHML. These results indicate that theHMLI silencer establishes a boundary between active and inactive chromatin atHML, but does so by organizing inactive chromatin in only one direction. This represents a different mechanism for delimiting the boundaries of a eukaryotic chromosome domain.
Postsynaptic clustering of γ-aminobutyric acid type A receptors by the γ3 subunit
in vivo
Authors: Baer Kristin , Essrich Christian , Benson Jack A. , Benke Dietmar , Bluethmann Horst , Fritschy Jean-Marc , Lüscher Bernhard
Published: July 2002

Synaptic localization of γ-aminobutyric acid type A (GABA
A
) receptors is a prerequisite for synaptic inhibitory function, but the mechanism by which different receptor subtypes are localized to postsynaptic sites is poorly understood. The γ2 subunit and the postsynaptic clustering protein gephyrin are required for synaptic localization and function of major GABA
A
receptor subtypes. We now show that transgenic overexpression of the γ3 subunit in γ2 subunit-deficient mice restores benzodiazepine binding sites, benzodiazepine-modulated whole cell currents, and postsynaptic miniature currents, suggesting the formation of functional, postsynaptic receptors. Moreover, the γ3 subunit can substitute for γ2 in the formation of GABA
A
receptors that are synaptically clustered and colocalized with gephyrin
in vivo
. These clusters were formed even in brain regions devoid of endogenous γ3 subunit, indicating that the factors present for clustering of γ2 subunit-containing receptors are sufficient to cluster γ3 subunit-containing receptors. The GABA
A
receptor and gephyrin-clustering properties of the ectopic γ3 subunit were also observed for the endogenous γ3 subunit, but only in the absence of the γ2 subunit, suggesting that the γ3 subunit is at a competitive disadvantage with the γ2 subunit for clustering of postsynaptic GABA
A
receptors in wild-type mice.
The role of the synthetic enzyme GAD65 in the control of neuronal γ-aminobutyric acid release
Authors: Tian Ning , Petersen Carl , Kash Shera , Baekkeskov Steinunn , Copenhagen David , Nicoll Roger
Published: July 2002

We have studied GABAergic synaptic transmission in retinal ganglion cells and hippocampal pyramidal cells to determine, at a cellular level, what is the effect of the targeted disruption of the gene encoding the synthetic enzyme GAD65 on the synaptic release of γ-aminobutyric acid (GABA). Neither the size nor the frequency of GABA-mediated spontaneous inhibitory postsynaptic currents (IPSCs) were reduced in retina or hippocampus in GAD65−/− mice. However, the release of GABA during sustained synaptic activation was substantially reduced. In the retina both electrical- and K
+
-induced increases in IPSC frequency were depressed without a change in IPSC amplitude. In the hippocampus the transient increase in the probability of inhibitory transmitter release associated with posttetanic potentiation was absent in the GAD65−/− mice. These results indicate that during and immediately after sustained stimulation the increase in the probability of transmitter release is not maintained in GAD65−/− mice. Such a finding suggests a decrease in the size or refilling kinetics of the releasable pool of vesicles, and various mechanisms are discussed that could account for such a defect.
Plasminogen deficiency leads to impaired remodeling after a toxic injury to the liver
Authors: Bezerra Jorge A. , Bugge Thomas H. , Melin-Aldana Hector , Sabla Gregg , Kombrinck Keith W. , Witte David P. , Degen Jay L.
Published: July 2002

Cellular proliferation and tissue remodeling are central to the regenerative response after a toxic injury to the liver. To explore the role of plasminogen in hepatic tissue remodeling and regeneration, we used carbon tetrachloride to induce an acute liver injury in plasminogen-deficient (Plg
o
) mice and nontransgenic littermates (Plg
+
). On day 2 after CCl
4
, livers of Plg
+
and Plg
o
mice had a similar diseased pale/lacy appearance, followed by restoration of normal appearance in Plg
+
livers by day 7. In contrast, Plg
o
livers remained diseased for as long as 2.5 months, with a diffuse pale/lacy appearance and persistent damage to centrilobular hepatocytes. The persistent centrilobular lesions were not a consequence of impaired proliferative response in Plg
o
mice. Notably, fibrin deposition was a prominent feature in diseased centrilobular areas in Plg
o
livers for at least 30 days after injury. Nonetheless, the genetically superimposed loss of the Aα fibrinogen chain (Plg
o
/Fib
o
mice) did not correct the abnormal phenotype. These data show that plasminogen deficiency impedes the clearance of necrotic tissue from a diseased hepatic microenvironment and the subsequent reconstitution of normal liver architecture in a fashion that is unrelated to circulating fibrinogen.
Toward controlling gene expression at will: Selection and design of zinc finger domains recognizing each of the 5′-GNN-3′ DNA target sequences
Authors: Segal David J. , Dreier Birgit , Beerli Roger R. , Barbas Carlos F.
Published: July 2002

We have taken a comprehensive approach to the generation of novel DNA binding zinc finger domains of defined specificity. Herein we describe the generation and characterization of a family of zinc finger domains developed for the recognition of each of the 16 possible 3-bp DNA binding sites having the sequence 5′-GNN-3′. Phage display libraries of zinc finger proteins were created and selected under conditions that favor enrichment of sequence-specific proteins. Zinc finger domains recognizing a number of sequences required refinement by site-directed mutagenesis that was guided by both phage selection data and structural information. In many cases, residues not expected to make base-specific contacts had effects on specificity. A number of these domains demonstrate exquisite specificity and discriminate between sequences that differ by a single base with >100-fold loss in affinity. We conclude that the three helical positions −1, 3, and 6 of a zinc finger domain are insufficient to allow for the fine specificity of the DNA binding domain to be predicted. These domains are functionally modular and may be recombined with one another to create polydactyl proteins capable of binding 18-bp sequences with subnanomolar affinity. The family of zinc finger domains described here is sufficient for the construction of 17 million novel proteins that bind the 5′-(GNN)
6
-3′ family of DNA sequences. These materials and methods should allow for the rapid construction of novel gene switches and provide the basis for a universal system for gene control.
Synthetic zeolites and other microporous oxide molecular sieves
Authors: Sherman John D.
Published: July 2002

Use of synthetic zeolites and other microporous oxides since
1950 has improved insulated windows, automobile air-conditioning,
refrigerators, air brakes on trucks, laundry detergents, etc. Their
large internal pore volumes, molecular-size pores, regularity of
crystal structures, and the diverse framework chemical compositions
allow “tailoring” of structure and properties. Thus, highly
active and selective catalysts as well as adsorbents and ion exchangers
with high capacities and selectivities were developed. In the petroleum
refining and petrochemical industries, zeolites have made possible
cheaper and lead-free gasoline, higher performance and lower-cost
synthetic fibers and plastics, and many improvements in process
efficiency and quality and in performance. Zeolites also help protect
the environment by improving energy efficiency, reducing automobile
exhaust and other emissions, cleaning up hazardous wastes (including
the Three Mile Island nuclear power plant and other radioactive
wastes), and, as specially tailored desiccants, facilitating the
substitution of new refrigerants for the ozone-depleting
chlorofluorocarbons banned by the Montreal Protocol.