bluuebunny/crossref_metadata_2025 huggingface dataset:
Interhemispheric disconnection syndrome in Alzheimer’s disease
Authors: Lakmache Yamina , Lassonde Maryse , Gauthier Serge , Frigon Jean-Yves , Lepore Franco
Published: July 2002

It is commonly acknowledged that patients with Alzheimer’s disease show memory and cognitive deficits that result from their cerebral histopathological abnormalities. We report new evidence showing that they also manifest deficits in interhemispheric integration of information, probably reflecting a corpus callosum dysfunction. Patients were given a battery of motor, somatosensory, and visual tests that had to be carried out by using either one or both hemispheres. Tasks were chosen such that subjects with Alzheimer’s disease performed normally when using intrahemispheric processing. They, however, performed poorly when interhemispheric communication was required. This observation attests to the presence of a disconnection syndrome and suggests that these interhemispheric tasks can serve as diagnostic tools for the early assessment of their dementia.
Base pair switching by interconversion of sugar puckers in DNA extended by proteins of RecA-family: A model for homology search in homologous genetic recombination
Authors: Nishinaka Taro , Shinohara Akira , Ito Yutaka , Yokoyama Shigeyuki , Shibata Takehiko
Published: July 2002

Escherichia coli
RecA is a representative of proteins from the RecA family, which promote homologous pairing and strand exchange between double-stranded DNA and single-stranded DNA. These reactions are essential for homologous genetic recombination in various organisms. From NMR studies, we previously reported a novel deoxyribose-base stacking interaction between adjacent residues on the extended single-stranded DNA bound to RecA protein. In this study, we found that the same DNA structure was induced by the binding to
Saccharomyces cerevisiae
Rad51 protein, indicating that the unique DNA structure induced by the binding to RecA-homologs was conserved from prokaryotes to eukaryotes. On the basis of this structure, we have formulated the structure of duplex DNA within filaments formed by RecA protein and its homologs. Two types of molecular structures are presented. One is the duplex structure that has the N-type sugar pucker. Its helical pitch is ≈95 Å (18.6 bp/turn), corresponding to that of an active, or ATP-form of the RecA filament. The other is one that has the S-type sugar pucker. Its helical pitch is ≈64 Å (12.5 bp/turn), corresponding to that of an inactive, or ADP-form of the RecA filament. During this modeling, we found that the interconversion of sugar puckers between the N-type and the S-type rotates bases horizontally, while maintaining the deoxyribose-base stacking interaction. We propose that this base rotation enables base pair switching between double-stranded DNA and single-stranded DNA to take place, facilitating homologous pairing and strand exchange. A possible mechanism for strand exchange involving DNA rotation also is discussed.
CC-chemokines enhance the replication of T-tropic strains of HIV-1 in CD4+T Cells: Role of signal transduction
Authors: Kinter Audrey , Catanzaro Andrew , Monaco JoAn , Ruiz Margaret , Justement Jesse , Moir Susan , Arthos James , Oliva Alessandra , Ehler Linda , Mizell Stephanie , Jackson Robert , Ostrowski Mario , Hoxie James , Offord Robin , Fauci Anthony S.
Published: July 2002

This study demonstrates that several CC-chemokines, including those that inhibit entry and replication of macrophage-tropic strains of HIV, increase the replication of T cell (T)-tropic strains in CD4+T cells. Enhancement of T-tropic HIV replication is observed at early stages of replication, requires signaling through inhibitory guanine nucleotide-binding regulatory (Gi) proteins, and is associated with increased cell surface colocalization of CD4 and the T-tropic HIV coreceptor CXCR4. These findings may further our understanding of the factors that influence the replication and spread of T-tropic strains of HIVin vivoand suggest that the use of cell signaling CC-chemokines as therapeutic agents for the purpose of limiting HIV replicationin vivoshould be approached with caution.
Patch-clamp and amperometric recordings from norepinephrine transporters: Channel activity and voltage-dependent uptake
Authors: Galli Aurelio , Blakely Randy D. , DeFelice Louis J.
Published: July 2002

Transporters for the biogenic amines dopamine, norepinephrine, epinephrine and serotonin are largely responsible for transmitter inactivation after release. They also serve as high-affinity targets for a number of clinically relevant psychoactive agents, including antidepressants, cocaine, and amphetamines. Despite their prominent role in neurotransmitter inactivation and drug responses, we lack a clear understanding of the permeation pathway or regulation mechanisms at the single transporter level. The resolution of radiotracer-based flux techniques limits the opportunities to dissect these problems. Here we combine patch-clamp recording techniques with microamperometry to record the transporter-mediated flux of norepinephrine across isolated membrane patches. These data reveal voltage-dependent norepinephrine flux that correlates temporally with antidepressant-sensitive transporter currents in the same patch. Furthermore, we resolve unitary flux events linked with bursts of transporter channel openings. These findings indicate that norepinephrine transporters are capable of transporting neurotransmitter across the membrane in discrete shots containing hundreds of molecules. Amperometry is used widely to study neurotransmitter distribution and kinetics in the nervous system and to detect transmitter release during vesicular exocytosis. Of interest regarding the present application is the use of amperometry on inside-out patches with synchronous recording of flux and current. Thus, our results further demonstrate a powerful method to assess transporter function and regulation.
A transmembrane form of annexin XII detected by site-directed spin labeling
Authors: Langen Ralf , Isas J. Mario , Hubbell Wayne L. , Haigler Harry T.
Published: July 2002

Previous studies of the annexin family of Ca
2+
binding proteins identified a soluble monomer in the absence of Ca
2+
and a trimer adsorbed on the membrane surface in the presence of Ca
2+
. On the basis of site-directed spin-labeling studies of annexin XII at low pH, we now report a membrane-inserted form of the protein with a dramatically different structure. The data suggest that upon insertion a continuous transmembrane α-helix is reversibly formed from a helix–loop–helix motif in the solution structure. Other regions with similar membrane-insertion potential were identified in the amino acid sequence, and we propose that the corresponding helices come together to form an aqueous pore that mediates the ion channel activity reported for several annexins.
A model system to study genomic imprinting of human genes
Authors: Gabriel J. M. , Higgins M. J. , Gebuhr T. C. , Shows T. B. , Saitoh S. , Nicholls R. D.
Published: July 2002

Somatic-cell hybrids have been shown to maintain the correct epigenetic chromatin states to study developmental globin gene expression as well as gene expression on the active and inactive X chromosomes. This suggests the potential use of somatic-cell hybrids containing either a maternal or a paternal human chromosome as a model system to study known imprinted genes and to identify as-yet-unknown imprinted genes. Testing gene expression by using reverse transcription followed by PCR, we show that functional imprints are maintained at four previously characterized 15q11–q13 loci in hybrids containing a single human chromosome 15 and at two chromosome 11p15 loci in hybrids containing a single chromosome 11. In contrast, three γ-aminobutyric acid type A receptor subunit genes in 15q12–q13 are nonimprinted. Furthermore, we have found that differential DNA methylation imprints at the
SNRPN
promoter and at a CpG island in 11p15 are also maintained in somatic-cell hybrids. Somatic-cell hybrids therefore are a valid and powerful system for studying known imprinted genes as well as for rapidly identifying new imprinted genes.
Stimulation of bacteriophage T4 middle transcription by the T4 proteins MotA and AsiA occurs at two distinct steps in the transcription cycle
Authors: Adelman Karen , Brody Edward N. , Buckle Malcolm
Published: July 2002

The bacteriophage T4 encodes proteins that are responsible for tightly regulating mRNA synthesis throughout phage development in
Escherichia coli
. The three classes of T4 promoters (early, middle, and late) are utilized sequentially by the host RNA polymerase as a result of phage-induced modifications. One such modification is the tight binding of the T4 AsiA protein to the σ
70
subunit of the RNA polymerase. This interaction is pivotal for the transition between T4 early and middle transcription, since it both inhibits recognition of host and T4 early promoters and stimulates T4 middle mode synthesis. The activation of T4 middle transcription also requires the T4 MotA protein, bound specifically to its recognition sequence, the “Mot box,” which is centered at position −30 of these promoters. Accordingly, the two T4 proteins working in concert are sufficient to effectively switch the transcription specificity of the RNA polymerase holoenzyme. Herein, we investigate the mechanism of transcription activation and report that, while the presence of MotA and AsiA increases the initial recruitment of RNA polymerase to a T4 middle promoter, it does not alter the intrinsic stability of the discrete complexes formed. In addition, we have characterized the RNA polymerase-promoter species by UV laser footprinting and followed their evolution from open into initiating complexes. These data, combined with
in vitro
transcription assays, indicate that AsiA and MotA facilitate promoter escape, thereby stimulating the production of full-length transcripts.
Antisense inhibition of the PTI-1 oncogene reverses cancer phenotypes
Authors: Su Zao-zhong , Goldstein Neil I. , Fisher Paul B.
Published: July 2002

The genetic alterations and molecular events mediating human prostate cancer development and progression remain to be defined. Rapid expression cloning and differential RNA display detect a putative oncogene, prostate tumor-inducing gene 1 (PTI-1), that is differentially expressed in human prostate (as well as breast, colon, and small cell lung) cancer cell lines, patient-derived prostate carcinomas, and blood from patients with metastatic prostate cancer. PTI-1 consists of a unique 5′ untranslated region (5′ UTR) with significant sequence homology to
Mycoplasma hyopneumoniae
23S ribosomal RNA juxtaposed to a sequence that encodes a truncated and mutated human elongation factor 1α (Trun-EF). Stable expression of a nearly full-length 1.9-kb PTI-1 gene, but not the separate PTI-1 5′ UTR or Trun-EF region, in normal rat embryo fibroblast cells, CREF-Trans 6, induces an aggressive tumorigenic phenotype in athymic nude mice. Blocking PTI-1 expression with antisense PTI-1 results in reversion of transformed PTI-1-expressing cells to a more normal cellular morphology with suppression in both anchorage-independent growth and tumorigenic potential in athymic nude mice. These findings document that PTI-1 is indeed an oncogene, and directly blocking PTI-1 expression can nullify cancer phenotypes. In these contexts, PTI-1 not only represents a gene with discriminating diagnostic properties but also may serve as a target for the gene-based therapy of human prostate and other cancers.
Homeobox genes in the ribbonwormLineus sanguineus: Evolutionary implications
Authors: Kmita-Cunisse Marie , Loosli Felix , Bièrne Jacques , Gehring Walter J.
Published: July 2002

From our current understanding of the genetic basis of development and pattern formation inDrosophilaand vertebrates it is commonly thought that clusters ofHoxgenes sculpt the morphology of animals in specific body regions. Based onHoxgene conservation throughout the animal kingdom it is proposed that these genes and their role in pattern formation evolved early during the evolution of metazoans. Knowledge of the history ofHoxgenes will lead to a better understanding of the role ofHoxgenes in the evolution of animal body plans. To inferHoxgene evolution, reliable data on lower chordates and invertebrates are crucial. Among the lower triploblasts, the body plan of the ribbonwormLineus(nemertini) appears to be close to the common ancestral condition of protostomes and deuterostomes. In this paper we present the isolation and identification ofHoxgenes inLineus sanguineus. We find that theLineusgenome contains a single cluster of at least sixHoxgenes: two anterior-class genes, three middle-class genes, and one posterior-class gene. Each of the genes can be definitely assigned to an ortholog group on the basis of its homeobox and its flanking sequences. The most closely related homeodomain sequences are invariably found among the mouse or Amphioxus orthologs, rather thanDrosophilaand other invertebrates. This suggests that the ribbonworms have diverged relatively little from the last common ancestors of protostomes and deuterostomes, the urbilateria.
Identification of cytokeratin 1 as a binding protein and presentation receptor for kininogens on endothelial cells
Authors: Hasan Ahmed A. K. , Zisman Timothy , Schmaier Alvin H.
Published: July 2002

A kininogen binding protein(s), a putative receptor, was identified on endothelial cells. A 54-kDa protein was isolated by a biotin–high molecular mass kininogen (HK) affinity column that, on aminoterminal sequencing of tryptic digests, was identified as cytokeratin 1. Multiple antibodies directed to cytokeratin 1 reacted with a 54-kDa band on immunoblot of lysates of endothelial cells. On laser scanning confocal microscopy, cytokeratin 1 antigen was found on the surface of endothelial cells. Cytokeratin 1 antigen also was detected on endothelial cell membranes by flow cytometry. Moreover, an antipeptide antibody to a sequence unique to cytokeratin 1 also specifically bound to nonpermeabilized endothelial cells. Biotin–HK specifically bound to cytokeratin only in the presence of Zn2+, and cytokeratin blocked biotin–HK binding to endothelial cells. Further, HK and low molecular mass kininogen, but not factor XII, blocked biotin–HK binding to cytokeratin, and peptides of each cell binding region of HK on domains 3,4, and 5 blocked biotin–HK binding to cytokeratin. gC1qR and soluble urokinase-like plasminogen activator receptor also inhibited biotin–HK binding to cytokeratin. These investigations identify a new function for cytokeratin 1 as a kininogen binding protein. Cytokeratins, members of the family of intermediate filament proteins, may represent a new class of receptors.
Perturbation of nucleosome core structure by the SWI/SNF complex persists after its detachment, enhancing subsequent transcription factor binding
Authors: Côté Jacques , Peterson Craig L. , Workman Jerry L.
Published: July 2002

To investigate the mechanism of SWI/SNF action, we have analyzed the pathway by which SWI/SNF stimulates formation of transcription factor-bound nucleosome core complexes. We report here that the SWI/SNF complex binds directly to nucleosome cores and uses the energy of ATP hydrolysis to disrupt histone/DNA interactions, altering the preferred path of DNA bending around the histone octamer. This disruption occurs without dissociating the DNA from the surface of the histone octamer. ATP-dependent disruption of nucleosomal DNA by SWI/SNF generates an altered nucleosome core conformation that can persist for an extended period after detachment of the SWI/SNF complex. This disrupted conformation retains an enhanced affinity for the transcription factor GAL4-AH. Thus, ATP-dependent nucleosome core disruption and enhanced binding of the transcription factor can be temporally separated. These results indicate that SWI/SNF can act transiently in the remodeling of chromatin structure, even before interactions of transcription factors.
Cdc20 associates with the kinase aurora2/Aik
Authors: Farruggio Dawn C. , Townsley Fiona M. , Ruderman Joan V.
Published: July 2002

Cdc20/fizzy family proteins are involved in activation of the anaphase-promoting complex/cyclosome, which catalyzes the ubiquitin-dependent proteolysis of cell cycle regulatory proteins such as anaphase inhibitors and mitotic cyclins, leading to chromosome segregation and exit from mitosis. Previous work has shown that human Cdc20 (hCdc20/p55CDC) associates with one or more kinases. We report here that Cdc20-associated myelin basic protein kinase activity peaks sharply in early M phase (embryonic cells) or in G
2
phase (somatic cells). In HeLa cells, Cdc20 is associated with the kinase aurora2/Aik. Aurora2/Aik is a member of the aurora/Ipl1 family of kinases that, like Cdc20, previously has been shown to be localized at mitotic spindle poles and is involved in regulating chromosome segregation and maintaining genomic stability. The demonstration that Cdc20 is associated with aurora2/Aik suggests that some function of Cdc20 is carried out or regulated through its association with aurora2/Aik.
Conditional requirement for the Flk-1 receptor in the
in vitro
generation of early hematopoietic cells
Authors: Hidaka Michihiro , Stanford William L. , Bernstein Alan
Published: July 2002

Genetic studies in mice have previously demonstrated an intrinsic requirement for the vascular endothelial growth factor (VEGF) receptor Flk-1 in the early development of both the hematopoietic and endothelial cell lineages. In this study, embryonic stem (ES) cells homozygous for a targeted null mutation in
flk-1
(
flk-1
(−/−)) were examined for their hematopoietic potential
in vitro
during embryoid body (EB) formation or when cultured on the stromal cell line OP9. Surprisingly, in EB cultures
flk-1
(−/−) ES cells were able to differentiate into all myeloid-erythroid lineages, albeit at half the frequency of heterozygous lines. In contrast, although
flk-1
(−/−) ES cells formed mesodermal-like colonies on OP9 monolayers, they failed to generate hematopoietic clusters even in the presence of exogenous cytokines. However,
flk-1
(−/−) OP9 cultures did contain myeloid precursors, albeit at greatly reduced percentages. This defect was rescued by first allowing
flk-1
(−/−) ES cells to differentiate into EBs and then passaging these cells onto OP9 stroma. Thus, the requirement for Flk-1 in early hematopoietic development can be abrogated by alterations in the microenvironment. This finding is consistent with a role for Flk-1 in regulating the migration of early mesodermally derived precursors into a microenvironment that is permissive for hematopoiesis.
p200 ARF–GEP1: A Golgi-localized guanine nucleotide exchange protein whose Sec7 domain is targeted by the drug brefeldin A
Authors: Mansour Sam J. , Skaug Jennifer , Zhao Xin-Hua , Giordano Jennifer , Scherer Stephen W. , Melançon Paul
Published: July 2002

The drug brefeldin A (BFA) disrupts protein traffic and Golgi morphology by blocking activation of ADP ribosylation factors (ARFs) through an unknown mechanism. Here, we investigated the cellular localization and BFA sensitivity of human
p200 ARF-GEP1
(
p200
), a ubiquitously expressed guanine nucleotide exchange factor of the Sec7 domain family. Multiple tagged forms of the full-length polypeptide localized to tight ribbon-like perinuclear structures that overlapped with the Golgi marker mannosidase II and were distinct from the pattern observed with ERGIC53/58. Analysis of several truncated forms mapped the Golgi-localization signal to the N-terminal third of p200. BFA treatment of transiently or stably transfected cells resulted in the redistribution of Golgi markers and in loss of cell viability, thereby indicating that overproduction of p200 may not be sufficient to overcome the toxic effect. A 39-kDa fragment spanning the Sec7 domain catalyzed loading of guanosine 5′-[γ-thio]triphosphate onto class I ARFs and displayed clear sensitivity to BFA. Kinetic analysis established that BFA did not compete with ARF for interaction with p200 but, rather, acted as an uncompetitive inhibitor that only targeted the p200-ARF complex with an inhibition constant of 7 μM. On the basis of these results, we propose that accumulation of an abortive p200-ARF complex in the presence of BFA likely leads to disruption of Golgi morphology.
p200
mapped to chromosome 8q13, 3.56 centirays from WI-6151, and database searches revealed the presence of putative isoforms whose inhibition may account for the effects of BFA on various organelles.
A single amino acid substitution in the cyclin D binding domain of the infected cell protein no. 0 abrogates the neuroinvasiveness of herpes simplex virus without affecting its ability to replicate
Authors: Van Sant Charles , Kawaguchi Yasushi , Roizman Bernard
Published: July 2002

The infected cell protein no. 0 (ICP0) of herpes simplex virus 1 is a promiscuous transactivator shown to enhance the expression of genes introduced into cells by infection or transfection. The protein interacts with several viral and cellular proteins. Earlier studies have shown that ICP0 binds and stabilizes cyclin D3 but does interfere with the phosphorylation of retinoblastoma protein, its major function. Cyclin D3 plays a key role in the transition from G
1
to S phase. To define the role of cyclin D3 in productive infection, the ICP0 binding site for cyclin D3 was mapped and mutagenized by substitution of aspartic acid codon 199 with the alanine codon. We report that the substitution precluded the interaction of this protein with cyclin D3 in the yeast two-hybrid system and the stabilization of cyclin D3 in infected cells. A recombinant virus carrying this mutation could not be differentiated from wild-type parent with respect to replication in dividing cells but yielded 10-fold less progeny from infected resting cells and serum-deprived or contact-inhibited human fibroblasts. In mice, the mutant was only slightly less pathogenic than the wild-type parent by intracerebral route but was significantly less neuroinvasive after peripheral inoculation. Replacement of the mutated amino acid with aspartic acid restored wild-type phenotype. Stabilization of cyclin D3 therefore is linked to higher virus yields in nondividing cells and potentially higher virulence in experimental and natural hosts. One function of ICP0 is to scavenge the cell for proteins that could bolster viral replication.
Cubilin, the endocytic receptor for intrinsic factor-vitamin B
12
complex, mediates high-density lipoprotein holoparticle endocytosis
Authors: Hammad Samar M. , Stefansson Steingrimur , Twal Waleed O. , Drake Christopher J. , Fleming Paul , Remaley Alan , Brewer H. Bryan , Argraves W. Scott
Published: July 2002

Receptors that endocytose high-density lipoproteins (HDL) have been elusive. Here yolk-sac endoderm-like cells were used to identify an endocytic receptor for HDL. The receptor was isolated by HDL affinity chromatography and identified as cubilin, the recently described endocytic receptor for intrinsic factor-vitamin B
12
. Cubilin antibodies inhibit HDL endocytosis by the endoderm-like cells and in mouse embryo yolk-sac endoderm, a prominent site of cubilin expression. Cubilin-mediated HDL endocytosis is inhibitable by HDL
2
, HDL
3
, apolipoprotein (apo)A-I, apoA-II, apoE, and RAP, but not by low-density lipoprotein (LDL), oxidized LDL, VLDL, apoC-I, apoC-III, or heparin. These findings, coupled with the fact that cubilin is expressed in kidney proximal tubules, suggest a role for this receptor in embryonic acquisition of maternal HDL and renal catabolism of filterable forms of HDL.
Translocation and specific cleavage of bacteriophage T7 DNA
in vivo
by
Eco
KI
Authors: García L. René , Molineux Ian J.
Published: July 2002

Infection of
Escherichia coli
containing the type I restriction enzyme
Eco
KI by bacteriophage T7
0.3
mutants leads to restriction during the late stages of genome entry and during DNA replication. Patterns of cleavage
in vivo
suggest that some cutting occurs near the midpoint of two recognition sites, consistent with the idea that
Eco
KI translocates DNA bidirectionally through itself and cuts when two enzyme molecules collide. Rapid ejection of a
0.3
+
T7 genome from a bacteriophage λ particle results in degradation of the infecting DNA by
Eco
KI, showing that the normal T7 DNA translocation process delays restriction. A unique recognition site inserted at the genomic left end allows
Eco
KI to function as a molecular motor and to translocate the remaining 39 kilobases of T7 DNA into the cell.
Glycosylation differences between the normal and pathogenic prion protein isoforms
Authors: Rudd Pauline M. , Endo Tama , Colominas Cristina , Groth Darlene , Wheeler Susan F. , Harvey David J. , Wormald Mark R. , Serban Hana , Prusiner Stanley B. , Kobata Akira , Dwek Raymond A.
Published: July 2002

Prion protein consists of an ensemble of glycosylated variants or
glycoforms. The enzymes that direct oligosaccharide processing, and
hence control the glycan profile for any given glycoprotein, are often
exquisitely sensitive to other events taking place within the cell in
which the glycoprotein is expressed. Alterations in the populations of
sugars attached to proteins can reflect changes caused, for example, by
developmental processes or by disease. Here we report that normal
(PrP
C
) and pathogenic (PrP
Sc
) prion proteins
(PrP) from Syrian hamsters contain the same set of at least 52 bi-,
tri-, and tetraantennary N-linked oligosaccharides, although the
relative proportions of individual glycans differ. This conservation of
structure suggests that the conversion of PrP
C
into
PrP
Sc
is not confined to a subset of PrPs that contain
specific sugars. Compared with PrP
C
, PrP
Sc
contains decreased levels of glycans with bisecting GlcNAc residues and
increased levels of tri- and tetraantennary sugars. This change is
consistent with a decrease in the activity of
N
-acetylglucosaminyltransferase III (GnTIII) toward
PrP
C
in cells where PrP
Sc
is formed and argues
that, in at least some cells forming PrP
Sc
, the
glycosylation machinery has been perturbed. The reduction in GnTIII
activity is intriguing both with respect to the pathogenesis of the
prion disease and the replication pathway for prions.
The single minichromosome maintenance protein of
Methanobacterium thermoautotrophicum
ΔH contains DNA helicase activity
Authors: Kelman Zvi , Lee Joon-Kyu , Hurwitz Jerard
Published: July 2002

Previous studies have identified an ATP-dependent DNA helicase activity intrinsic to the human minichromosome maintenance (MCM) complex, composed of MCM subunits 4, 6, and 7 [Ishimi, Y. (1997)
J. Biol. Chem.
272, 24508–24513]. In contrast to the presence of multiple MCM genes (at least six) in eukaryotes, the archaeon
Methanobacterium thermoautotrophicum
ΔH (mth) genome contains a single open reading frame coding for an MCM protein. In this study we report the isolation of the mthMCM protein overexpressed in
Escherichia coli
. The purified recombinant protein was found to exist in both multimeric (≈10
3
kDa) and monomeric (76 kDa) forms. Both forms of the protein bind to single-stranded DNA, hydrolyze ATP in the presence of DNA, and possess 3′-to-5′ ATP-dependent DNA helicase activities. Thus, a single mthMCM protein contains biochemical properties identical to those associated with the eukaryotic MCM4, -6, and -7 complex. These results suggest that the characterization of the mthMCM protein and its multiple forms may contribute to our understanding of the role of MCM helicase activity in eukaryotic chromosomal DNA replication.
Tolerance of Arc repressor to multiple-alanine substitutions
Authors: Brown Bronwen M. , Sauer Robert T.
Published: July 2002

Arc repressor mutants containing from three to 15 multiple-alanine substitutions have spectral properties expected for native Arc proteins, form heterodimers with wild-type Arc, denature cooperatively with
T
m
s equal to or greater than wild type, and, in some cases, fold as much as 30-fold faster and unfold as much as 50-fold slower than wild type. Two of the mutants, containing a total of 14 different substitutions, also footprint operator DNA
in vitro
. The stability of some of the proteins with multiple-alanine mutations is significantly greater than that predicted from the sum of the single substitutions, suggesting that a subset of the wild-type residues in Arc may interact in an unfavorable fashion. Overall, these results show that almost half of the residues in Arc can be replaced by alanine en masse without compromising the ability of this small, homodimeric protein to fold into a stable, native-like structure.
DNA replication in vertebrates requires a homolog of the Cdc7 protein kinase
Authors: Roberts B. Tibor , Ying Carol Y. , Gautier Jean , Maller James L.
Published: July 2002

CDC7
is an essential gene required for DNA replication in
Saccharomyces cerevisiae
. Cdc7p homologs have recently been identified in vertebrates, but their role in DNA replication has not yet been addressed. Here we show that antibodies to the
Xenopus laevis
homolog, xCdc7, interfere with DNA replication
in vivo
in developing embryos and
in vitro
in cycling egg extracts. We also demonstrate cell cycle-dependent association of xCdc7 with the Mcm complex, which binds to replication origins and also is required for DNA synthesis. Taken together, these data indicate that the function of xCdc7 is conserved from fungi to vertebrates. xCdc7 protein accumulates after stimulation of resting oocytes with progesterone, suggesting a molecular explanation for previous observations that the development of the capacity for DNA replication requires protein synthesis late in meiosis I.
Identification of a putative effector protein for rab11 that participates in transferrin recycling
Authors: Zeng Jianbo , Ren Mindong , Gravotta Diego , De Lemos-Chiarandini Carmen , Lui Mary , Erdjument-Bromage Hediye , Tempst Paul , Xu Genxing , Shen Tian Huai , Morimoto Takashi , Adesnik Milton , Sabatini David D.
Published: July 2002

We have identified and cloned the cDNA for a 912-aa protein, rab11BP, that interacts with the GTP-containing active form of rab11, a GTP-binding protein that plays a critical role in receptor recycling. Although rab11BP is primarily cytosolic, a significant fraction colocalizes with rab11 in endosomal membranes of both the sorting and recycling subcompartments.
In vitro
binding of rab11 to native rab11BP requires partial denaturation of the latter to expose an internal binding site located between residues 334 and 504 that is apparently masked by the C-terminal portion of the protein, which includes six repeats known as WD40 domains. Within the cell, rab11BP must undergo a conformational change in which the rab11-binding site becomes exposed, because when coexpressed with rab11 in transfected cells the two proteins formed abundant complexes in association with membranes. Furthermore, although overexpression of rab11BP did not affect transferrin recycling, overexpression of a truncated form of the protein, rab11BP(1–504), that includes the rab11-binding site but lacks the WD40 domains inhibited recycling as strongly as does a dominant negative rab11 mutant protein that does not bind GTP. Strikingly, the inhibition caused by the truncated rab11BP was prevented completely when the cells also expressed a C-terminally deleted, nonprenylatable form of rab11 that, by itself, has no effect on recycling. We propose that rab11BP is an effector for rab11, whose association with this GTP-binding protein is dependent on the action of another membrane-associated factor that promotes the unmasking of the rab11-binding site in rab11BP.
NMR and mutagenesis evidence for an I domain allosteric site that regulates lymphocyte function-associated antigen 1 ligand binding
Authors: Huth Jeffrey R. , Olejniczak Edward T. , Mendoza Renaldo , Liang Heng , Harris Edith A. S. , Lupher Mark L. , Wilson Amy E. , Fesik Stephen W. , Staunton Donald E.
Published: July 2002

The leukocyte integrin, lymphocyte function-associated antigen 1 (LFA-1) (CD11a/CD18), mediates cell adhesion and signaling in inflammatory and immune responses. To support these functions, LFA-1 must convert from a resting to an activated state that avidly binds its ligands such as intercellular adhesion molecule 1 (ICAM-1). Biochemical and x-ray studies of the Mac-1 (CD11b/CD18) I domain suggest that integrin activation could involve a conformational change of the C-terminal α-helix. We report the use of NMR spectroscopy to identify CD11a I domain residues whose resonances are affected by binding to ICAM-1. We observed two distinct sites in the CD11a I domain that were affected. As expected from previous mutagenesis studies, a cluster of residues localized around the metal ion-dependent adhesion site (MIDAS) was severely perturbed on ICAM-1 binding. A second cluster of residues distal to the MIDAS that included the C-terminal α-helix of the CD11a I domain was also affected. Substitution of residues in the core of this second I domain site resulted in constitutively active LFA-1 binding to ICAM-1. Binding data indicates that none of the 20 substitution mutants we tested at this second site form an essential ICAM-1 binding interface. We also demonstrate that residues in the I domain linker sequences can regulate LFA-1 binding. These results indicate that LFA-1 binding to ICAM-1 is regulated by an I domain allosteric site (IDAS) and that this site is structurally linked to the MIDAS.
The role of genetic and genomic attributes in the success of polyploids
Authors: Soltis Pamela S. , Soltis Douglas E.
Published: July 2002

In 1950, G. Ledyard Stebbins devoted two chapters of his book
Variation and Evolution in Plants
(Columbia Univ. Press,
New York) to polyploidy, one on occurrence and nature and one on
distribution and significance. Fifty years later, many of the questions
Stebbins posed have not been answered, and many new questions have
arisen. In this paper, we review some of the genetic attributes of
polyploids that have been suggested to account for the tremendous
success of polyploid plants. Based on a limited number of studies, we
conclude: (
i
) Polyploids, both individuals and
populations, generally maintain higher levels of heterozygosity than do
their diploid progenitors. (
ii
) Polyploids exhibit less
inbreeding depression than do their diploid parents and can therefore
tolerate higher levels of selfing; polyploid ferns indeed have higher
levels of selfing than do their diploid parents, but polyploid
angiosperms do not differ in outcrossing rates from their diploid
parents. (
iii
) Most polyploid species are polyphyletic,
having formed recurrently from genetically different diploid parents.
This mode of formation incorporates genetic diversity from multiple
progenitor populations into the polyploid “species”; thus,
genetic diversity in polyploid species is much higher than expected by
models of polyploid formation involving a single origin.
(
iv
) Genome rearrangement may be a common attribute of
polyploids, based on evidence from genome
in situ
hybridization (GISH), restriction fragment length polymorphism (RFLP)
analysis, and chromosome mapping. (
v
) Several groups of
plants may be ancient polyploids, with large regions of homologous DNA.
These duplicated genes and genomes can undergo divergent evolution and
evolve new functions. These genetic and genomic attributes of
polyploids may have both biochemical and ecological benefits that
contribute to the success of polyploids in nature.
Virus neutralization by germ-line vs. hypermutated antibodies
Authors: Kalinke Ulrich , Oxenius Annette , López-Macías Constantino , Zinkernagel Rolf M. , Hengartner Hans
Published: July 2002

Mice infected with vesicular stomatitis virus (VSV), a cytopathic virus closely related to rabies virus, mount a virus-neutralizing antibody response protecting against lethal disease. VSVneutralizing monoclonal IgGs isolated from primary immune responses were devoid of somatic mutations, whereas most secondary and all hyperimmune response IgGs tested were hypermutated. A comparative analysis of recombinant single-chain antibody fragments (scFv-Cκ) revealed that even the germ-line precursor of one hypermutated antibody bound and neutralized VSV. Four somatic amino acid substitutions in VHincreased by 300-fold the binding strength of monovalent scFv-Cκ. The multivalent binding avidity of germ-line scFv-Cκ was increased by more than 10-fold compared with the monovalent binding strength. In contrast, hypermutated scFv-Cκ did not show such avidity effects. Thus the overall binding difference between the germ-line and the hypermutated VSV-neutralizing antibody was only 10- to 15-fold. This may explain why primary germ-line antibodies and secondary hypermutated antibodies directed against pathogens such as viruses and bacteria expressing repetitive antibody determinants show rather similar binding qualities, whereas monovalently binding hapten-specific antibodies can show “affinity maturation” effects of up to 1000-fold.
Frequency of resistance to
Bacillus thuringiensis
in field populations of pink bollworm
Authors: Tabashnik Bruce E. , Patin Amanda L. , Dennehy Timothy J. , Liu Yong-Biao , Carrière Yves , Sims Maria A. , Antilla Larry
Published: July 2002

Strategies for delaying pest resistance to genetically modified
crops that produce
Bacillus thuringiensis
(Bt) toxins
are based primarily on theoretical models. One key assumption of such
models is that genes conferring resistance are rare. Previous estimates
for lepidopteran pests targeted by Bt crops seem to meet this
assumption. We report here that the estimated frequency of a recessive
allele conferring resistance to Bt toxin Cry1Ac was 0.16 (95%
confidence interval = 0.05–0.26) in strains of pink bollworm
(
Pectinophora gossypiella
) derived from 10 Arizona
cotton fields during 1997. Unexpectedly, the estimated resistance
allele frequency did not increase from 1997 to 1999 and Bt cotton
remained extremely effective against pink bollworm. These results
demonstrate that the assumptions and predictions of resistance
management models must be reexamined.
Calcium-dependent enzyme activation and vacuole formation in the apical granular region of pancreatic acinar cells
Authors: Raraty Michael , Ward Jeremy , Erdemli Gul , Vaillant Camille , Neoptolemos John P. , Sutton Robert , Petersen Ole H.
Published: July 2002

The pancreatic acinar cell produces powerful digestive enzymes
packaged in zymogen granules in the apical pole. Ca
2+
signals elicited by acetylcholine or cholecystokinin (CCK) initiate
enzyme secretion by exocytosis through the apical membrane.
Intracellular enzyme activation is normally kept to a minimum, but in
the often-fatal human disease acute pancreatitis, autodigestion occurs.
How the enzymes become inappropriately activated is unknown. We
monitored the cytosolic Ca
2+
concentration
([Ca
2+
]
i
), intracellular trypsin activation,
and its localization in isolated living cells with specific fluorescent
probes and studied intracellular vacuole formation by electron
microscopy as well as quantitative image analysis (light microscopy). A
physiological CCK level (10 pM) eliciting regular Ca
2+
spiking did not evoke intracellular trypsin activation or vacuole
formation. However, stimulation with 10 nM CCK, evoking a sustained
rise in [Ca
2+
]
i
, induced pronounced trypsin
activation and extensive vacuole formation, both localized in the
apical pole. Both processes were abolished by preventing abnormal
[Ca
2+
]
i
elevation, either by preincubation
with the specific Ca
2+
chelator
1,2-bis(O-aminophenoxy)ethane-N,N-N′,N′-tetraacetic acid (BAPTA) or by
removal of external Ca
2+
. CCK hyperstimulation evokes
intracellular trypsin activation and vacuole formation in the apical
granular pole. Both of these processes are mediated by an abnormal
sustained rise in [Ca
2+
]
i
.
τ kinases in the rat heat shock model: Possible implications for Alzheimer disease
Authors: Shanavas Alikunju , Papasozomenos Sozos Ch.
Published: July 2002

We have previously shown, by using the phosphate-dependent anti-τ
antibodies Tau-1 and PHF-1, that heat shock induces rapid
dephosphorylation of τ followed by hyperphosphorylation in female
rats. In this study, we analyzed in forebrain homogenates from female
Sprague–Dawley rats the activities of extracellular signal regulated
kinase 1/2 (ERK1/2), c-Jun NH
2
-terminal kinase (JNK),
glycogen synthase kinase-3β (GSK-3β), cyclin-dependent kinase 5
(Cdk5), cAMP-dependent protein kinase A (PKA), and
Ca
2+
/calmodulin-dependent protein kinase II
(CaMKII) at 0 (
n
= 5), 3 (
n
=
4), 6 (
n
= 5), and 12 (
n
= 5) h
after heat shock and in non-heat-shocked controls
(
n
= 5). Immunoprecipitation kinase assays at
0 h showed suppression of the activities of all kinases except of
GSK-3β, which showed increased activity. At 3–6 h, the activities of
ERK1/2, JNK, Cdk5, and GSK-3β toward selective substrates were
increased; however, only JNK, Cdk5, and GSK-3β but not ERK1/2 were
overactivated toward purified bovine τ. At 3–6 h, kinase assays
specific for PKA and CaMKII showed no increased activity toward either
τ or selective substrates. All of eight anti-τ antibodies tested
showed dephosphorylation at 0 h and hyperphosphorylation at 3–6
h, except for 12E8, which showed hyperphosphorylation also at 0 h.
Immunoblot analysis using activity-dependent antibodies against
ERK1/2, JNK, and GSK-3β confirmed the above data. Increased
activation and inhibition of kinases after heat shock were
statistically significant in comparison with controls. Because τ is
hyperphosphorylated in Alzheimer disease these findings suggest that
JNK, GSK-3β, and Cdk5 may play a role in its pathogenesis.
The Golgi-associated COPI-coated buds and vesicles contain β/γ-actin
Authors: Valderrama Ferran , Luna Ana , Babià Teresa , Martínez-Menárguez José A. , Ballesta José , Barth Holger , Chaponnier Christine , Renau-Piqueras Jaime , Egea Gustavo
Published: July 2002

It has been shown previously that the morphology and subcellular positioning of the Golgi complex is controlled by actin microfilaments. To further characterize the association between actin microfilaments and the Golgi complex, we have used theClostridium botulinumtoxins C2 and C3, which specifically inhibit actin polymerization and cause depolymerization of F-actin in intact cells by the ADP ribosylation of G-actin monomers and the Rho small GTP-binding protein, respectively. Normal rat kidney cells treated with C2 showed that disruption of the actin and the collapse of the Golgi complex occurred concomitantly. However, when cells were treated with C3, the actin disassembly was observed without any change in the organization of the Golgi complex. The absence of the involvement of Rho was further confirmed by the treatment with lysophosphatidic acid or microinjection with the constitutively activated form of RhoA, both of which induced the stress fiber formation without affecting the Golgi complex. Immunogold electron microscopy in normal rat kidney cells revealed that β- and γ-actin isoforms were found in Golgi-associated COPI-coated buds and vesicles. Taken together, the results suggest that the Rho signaling pathway does not directly regulate Golgi-associated actin microfilaments, and that β- and γ-actins might be involved in the formation and/or transport of Golgi-derived vesicular or tubular intermediates.
Validation française d'un inventaire de micro‐stresseurs de la vie quotidienne: combinaison du “Daily Hassles Scale” et du “Uplifts Scale”
Authors: Dumont Michelle , Tarabulsy George M. , Gagnon Jocelyne , Tessier Réjean , Provost Marc
Published: July 2002

Ce travail présente les résultats d'une étude de validité de la version française d'un instrument d'auto‐évaluation des micro‐stresseurs de la vie quotidienne (“Daily Hassles Scale”; “Uplifts Scale”; Lazarus & Folkman, 1989). Les informations de validité reposent sur des structures factorielles ainsi que des analyses de cohérence interne et de convergence avec une mesure de stress psychologique (MSP: Lemyre, Tessier, & Fillion, 1990). Auprès d'une communauté universitaire (N= 265), six facteurs d'embětements quotidiens et cinq facteurs d'encouragements journaliers ont été isolés. Comme prévu, le score d'intensité aux différents facteurs d'embětements corrèle positivement et significativement avec l'état d'ětre stressé ce qui n'est pas le cas des encouragements quotidiens. La discussion soulève la nécessité d'utiliser différents groupes de micro‐stresseurs plutôt qu'un facteur général. Elle évalue également le rôle ambigu des encouragements quotidiens comme facteur de protection dans l'expérience du stress.
Prescribing drug of choice to opiate dependent drug users: a comparison of clients receiving heroin with those receiving injectable methadone at a West London drug clinic
Authors: METREBIAN NICKY , SHANAHAN WILLIAM , STIMSON GERRY V. , SMALL COLIN , LEE MARK , MTUTU VICTOR , WELLS BRIAN
Published: July 2002

Fifty‐eight long‐term treatment resistant opiate‐dependent drug users were offered the choice of receiving injectable heroin or injectable methadone at a West London drug clinic. Drugs were dispensed on‐site at the clinic with weekend take‐home. There was no routine ongoing supervised injecting. A ceiling dose of 200mg/day of heroin or methadone was set. One‐third chose injectable methadone. Compared to those choosing heroin, these drug users were less likely to have used heroin or crack/cocaine before entering treatment, and were more likely to have previously received treatment with injectable methadone. Drug users reported choosing methadone because it was their primary drug of addiction, and compared with heroin has a longer duration of action and increased strength. Problems with each drug were reported: those choosing heroin complained that the upper dose limit was too low to maintain them adequately, and some receiving methadone complained of discomfort while injecting intravenously. While those choosing each drug had different baseline characteristics, both groups were well retained in treatment and at 3 months made significant reductions in drug use and crime, which were sustained over the 12‐month follow‐up period. There was no significant difference between treatment outcome between each group. There is an urgent need to conduct randomized controlled trials to establish the effectiveness of prescribing injectable methadone and heroin to inform policy and practice.
Factors related to self‐reported violent and accidental injuries
Authors: MACDONALD SCOTT , WELLS SAMANTHA
Published: July 2002

The main objective of this study is to gain a better understanding of factors that distinguish violent and accidental injuries. A secondary analysis was conducted on data from a randomized telephone survey of 10 385 Canadian residents. Three groups were compared using chi‐square tests and logistic regression analyses: respondents who reported no injuries in the previous year, those with at least one accidental injury and those with at least one violent injury. In the bivariate analyses, the violent injury group was significantly more likely than the accidental injury and non‐injury groups to be single, widowed, separated or divorced, have more than five drinks on a usual drinking occasion, experience harmful effects of alcohol and to have used illicit drugs, such as cocaine and marijuana, and licit drugs, such as antidepressants and sleeping pills. Finally, the violent injury group was significantly more likely than those with non‐violent injuries to report that the incident was related to either their own or someone else's alcohol or drug use. In the final multiple logistic regression analysis, variables significantly associated with injuries due to criminal victimizations compared with accidental injuries were being female, single, cocaine use of the injured and substance use of someone else during the injury.