bluuebunny/crossref_metadata_2025 huggingface dataset:
Free radical oxidation of brain proteins in accelerated senescence and its modulation by
N
-tert-butyl-α-phenylnitrone
Authors: Butterfield D. Allan , Howard Beverly J. , Yatin Servet , Allen Kerry L. , Carney John M.
Published: July 2002

According to the free radical theory of aging, reactive oxygen species cause oxidative damage, proposed to be an underlying factor of the aging process. In the current study, we have used electron paramagnetic resonance spin labeling, measurements of protein carbonyl content, an index of protein oxidation, and determination of the activity of glutamine synthetase (an oxidatively sensitive enzyme) to report that cortical synaptosomal membranes from the senescence accelerated-prone (SAMP8) mouse showed structural characteristics of free radical oxidative stress relative to the senescence accelerated-resistant (SAMR1) mouse. The SAMP8 mouse exhibited a decrease in the relevant EPR parameter consistent with oxidative stress (
P
< 0.002), a decreased glutamine synthetase activity (
P
< 0.05), and an increased protein carbonyl content (
P
< 0.01) compared with these parameters in the SAMR1 mouse. Further, because free radical trapping compounds have been demonstrated to extend maximum life span and improve cognition in SAMP8 mice, we investigated the protective nature of the known free radical scavenger,
N
-tert-butyl-α-phenylnitrone (PBN), on the physical state of cortical synaptosomal membrane proteins. For 14 days, SAMR1 and SAMP8 mice were injected with 30 mg/kg PBN while the controls were injected with the corresponding volume of saline. Characteristic of less oxidized systems, cortical synaptosomal membranes from the PBN-injected SAMP8 mouse exhibited a return toward normal values of the relevant EPR parameter [the M
I
= +1 low-field weakly immobilized line/M
I
= +1 low-field strongly immobilized line (W/S) ratio of a protein-specific spin label] (
P
< 0.001) compared with that from saline-injected SAMP8 mice. In SAMR1 mice, in contrast to SAMP8, there was no significant change in the conformation of membrane proteins or protein carbonyl content of cortical synaptosomal membranes from the PBN-injected and saline-injected SAMR1 mice, showing that PBN itself did not induce conformational changes in cortical synaptosomal membrane proteins. The results are discussed with reference to the use of free radical scavengers as potential anti-aging agents.
Inhibition of tumorigenesis by a cytosine–DNA, methyltransferase, antisense oligodeoxynucleotide
Authors: Ramchandani Shyam , MacLeod A. Robert , Pinard Marc , von Hofe Eric , Szyf Moshe
Published: July 2002

This paper tests the hypothesis that cytosine DNA methyltransferase (DNA MeTase) is a candidate target for anticancer therapy. Several observations have suggested recently that hyperactivation of DNA MeTase plays a critical role in initiation and progression of cancer and that its up-regulation is a component of the Ras oncogenic signaling pathway. We show that a phosphorothioate-modified, antisense oligodeoxynucleotide directed against the DNA MeTase mRNA reduces the level of DNA MeTase mRNA, inhibits DNA MeTase activity, and inhibits anchorage independent growth of Y1 adrenocortical carcinoma cells
ex vivo
in a dose-dependent manner. Injection of DNA MeTase antisense oligodeoxynucleotides i.p. inhibits the growth of Y1 tumors in syngeneic LAF1 mice, reduces the level of DNA MeTase, and induces demethylation of the adrenocortical-specific gene C21 and its expression in tumors
in vivo
. These results support the hypothesis that an increase in DNA MeTase activity is critical for tumorigenesis and is reversible by pharmacological inhibition of DNA MeTase.
Regulation of microtubule dynamics by the neuronal growth-associated protein SCG10
Authors: Riederer Beat M. , Pellier Véronique , Antonsson Bruno , Di Paolo Gilbert , Stimpson Stephen A. , Lütjens Robert , Catsicas Stefan , Grenningloh Gabriele
Published: July 2002

Dynamic assembly and disassembly of microtubules is essential for cell division, cell movements, and intracellular transport. In the developing nervous system, microtubule dynamics play a fundamental role during neurite outgrowth, elongation, and branching, but the molecular mechanisms involved are unknown. SCG10 is a neuron-specific protein that is membrane-associated and highly enriched in growth cones. Here we show that SCG10 binds to microtubules, inhibits their assembly, and can induce microtubule disassembly. We also show that SCG10 overexpression enhances neurite outgrowth in a stably transfected neuronal cell line. These data identify SCG10 as a key regulator of neurite extension through regulation of microtubule instability.
Structure at 2.7 Å resolution of the
Paracoccus denitrificans
two-subunit cytochrome
c
oxidase complexed with an antibody F
V
fragment
Authors: Ostermeier Christian , Harrenga Axel , Ermler Ulrich , Michel Hartmut
Published: July 2002

The
aa
3
type cytochrome
c
oxidase consisting of the core subunits I and II only was isolated from the soil bacterium
Paracoccus denitrificans
and crystallized as complex with a monoclonal antibody F
v
fragment. Crystals could be grown in the presence of a number of different nonionic detergents. However, only undecyl-β-
d
-maltoside and cyclohexyl-hexyl-β-
d
-maltoside yielded well-ordered crystals suitable for high resolution x-ray crystallographic studies. The crystals belong to space group P2
1
2
1
2
1
and diffract x-rays to at least 2.5 Å (1 Å = 0.1 nm) resolution using synchrotron radiation. The structure was determined to a resolution of 2.7 Å using molecular replacement and refined to a crystallographic
R
-factor of 20.5% (
R
free
= 25.9%). The refined model includes subunits I and II and the 2 chains of the F
v
fragment, 2 heme A molecules, 3 copper atoms, and 1 Mg/Mn atom, a new metal (Ca) binding site, 52 tentatively identified water molecules, and 9 detergent molecules. Only four of the water molecules are located in the cytoplasmic half of cytochrome
c
oxidase. Most of them are near the interface of subunits I and II. Several waters form a hydrogen-bonded cluster, including the heme propionates and the Mg/Mn binding site. The F
v
fragment binds to the periplasmic polar domain of subunit II and is critically involved in the formation of the crystal lattice. The crystallization procedure is well reproducible and will allow for the analysis of the structures of mechanistically interesting mutant cytochrome
c
oxidases.
Histones H3 and H4 are components of upstream activation factor required for the high-level transcription of yeast rDNA by RNA polymerase I
Authors: Keener John , Dodd Jonathan A. , Lalo Dominique , Nomura Masayasu
Published: July 2002

RNA polymerase I (Pol I) transcription in the yeast
Saccharomyces cerevisiae
is greatly stimulated
in vivo
and
in vitro
by the multiprotein complex, upstream activation factor (UAF). UAF binds tightly to the upstream element of the rDNA promoter, such that once bound (
in vitro
), UAF does not readily exchange onto a competing template. Of the polypeptides previously identified in purified UAF, three are encoded by genes required for Pol I transcription
in vivo
:
RRN5
,
RRN9
, and
RRN10
. Two others, p30 and p18, have remained uncharacterized. We report here that the N-terminal amino acid sequence, its mobility in gel electrophoresis, and the immunoreactivity of p18 shows that it is histone H3. In addition, histone H4 was found in UAF, and myc-tagged histone H4 could be used to affinity-purify UAF. Histones H2A and H2B were not detectable in UAF. These results suggest that histones H3 and H4 probably account for the strong binding of UAF to DNA and may offer a means by which general nuclear regulatory signals could be transmitted to Pol I.
Proliferation of adult T cell leukemia/lymphoma cells is associated with the constitutive activation of JAK/STAT proteins
Authors: Takemoto Shigeki , Mulloy James C. , Cereseto Anna , Migone Thi-Sao , Patel Barvin K. R. , Matsuoka Masao , Yamaguchi Kuzunari , Takatsuki Kiyoshi , Kamihira Shimeru , White Jeffrey D. , Leonard Warren J. , Waldmann Thomas , Franchini Genoveffa
Published: July 2002

Human T cell leukemia/lymphotropic virus type I (HTLV-I) induces adult T cell leukemia/lymphoma (ATLL). The mechanism of HTLV-I oncogenesis in T cells remains partly elusive.
In vitro
, HTLV-I induces ligand-independent transformation of human CD4
+
T cells, an event that correlates with acquisition of constitutive phosphorylation of Janus kinases (JAK) and signal transducers and activators of transcription (STAT) proteins. However, it is unclear whether the
in vitro
model of HTLV-I transformation has relevance to viral leukemogenesis
in vivo
. Here we tested the status of JAK/STAT phosphorylation and DNA-binding activity of STAT proteins in cell extracts of uncultured leukemic cells from 12 patients with ATLL by either DNA-binding assays, using DNA oligonucleotides specific for STAT-1 and STAT-3, STAT-5 and STAT-6 or, more directly, by immunoprecipitation and immunoblotting with anti-phosphotyrosine antibody for JAK and STAT proteins. Leukemic cells from 8 of 12 patients studied displayed constitutive DNA-binding activity of one or more STAT proteins, and the constitutive activation of the JAK/STAT pathway was found to persist over time in the 2 patients followed longitudinally. Furthermore, an association between JAK3 and STAT-1, STAT-3, and STAT-5 activation and cell-cycle progression was demonstrated by both propidium iodide staining and bromodeoxyuridine incorporation in cells of four patients tested. These results imply that JAK/STAT activation is associated with replication of leukemic cells and that therapeutic approaches aimed at JAK/STAT inhibition may be considered to halt neoplastic growth.
Interactive cloning with the SH3 domain of N-src identifies a new brain specific ion channel protein, with homology to Eag and cyclic nucleotide-gated channels
Authors: Santoro Bina , Grant Seth G. N. , Bartsch Dusan , Kandel Eric R.
Published: July 2002

We have isolated a novel cDNA, that appears to represent a new class of ion channels, by using the yeast two-hybrid system and the SH3 domain of the neural form of Src (N-src) as a bait. The encoded polypeptide, BCNG-1, is distantly related to cyclic nucleotide-gated channels and the voltage-gated channels, Eag and H-erg. BCNG-1 is expressed exclusively in the brain, as a glycosylated protein of ≈132 kDa. Immunohistochemical analysis indicates that BCNG-1 is preferentially expressed in specific subsets of neurons in the neocortex, hippocampus, and cerebellum, in particular pyramidal neurons and basket cells. Within individual neurons, the BCNG-1 protein is localized to either the dendrites or the axon terminals depending on the cell type. Southern blot analysis shows that several other BCNG-related sequences are present in the mouse genome, indicating the emergence of an entire subfamily of ion channel coding genes. These findings suggest the existence of a new type of ion channel, which is potentially able to modulate membrane excitability in the brain and could respond to regulation by cyclic nucleotides.
Bacterial infection as assessed by
in vivo
gene expression
Authors: Heithoff Douglas M. , Conner Christopher P. , Hanna Philip C. , Julio Steven M. , Hentschel Ute , Mahan Michael J.
Published: July 2002

In vivo e
xpression
t
echnology (IVET) has been used to identify >100
Salmonella typhimurium
genes that are specifically expressed during infection of BALB/c mice and/or murine cultured macrophages. Induction of these genes is shown to be required for survival in the animal under conditions of the IVET selection. One class of
in vivo i
nduced (
ivi
) genes,
iviVI-A
and
iviVI-B
, constitute an operon that resides in a region of the
Salmonella
genome with low G+C content and presumably has been acquired by horizontal transfer. These
ivi
genes encode predicted proteins that are similar to adhesins and invasins from prokaryotic and eukaryotic pathogens (
Escherichia coli
[
tia
],
Plasmodium falciparum
[PfEMP1]) and have coopted the PhoPQ regulatory circuitry of
Salmonella
virulence genes. Examination of the
in vivo
induction profile indicates (
i
) many
ivi
genes encode regulatory functions (e.g.,
phoPQ
and
pmrAB
) that serve to enhance the sensitivity and amplitude of virulence gene expression (e.g.,
spvB
); (
ii
) the biochemical function of many metabolic genes may not represent their sole contribution to virulence; (
iii
) the host ecology can be inferred from the biochemical functions of
ivi
genes; and (
iv
) nutrient limitation plays a dual signaling role in pathogenesis: to induce metabolic functions that complement host nutritional deficiencies and to induce virulence functions required for immediate survival and spread to subsequent host sites.
Ataxia and altered dendritic calcium signaling in mice carrying a targeted null mutation of the calbindin D28k gene
Authors: Airaksinen Matti S. , Eilers Jens , Garaschuk Olga , Thoenen Hans , Konnerth Arthur , Meyer Michael
Published: July 2002

Intracellular calcium-binding proteins are abundantly expressed in many neuronal populations. Previous evidence suggests that calcium-binding proteins can modulate various neuronal properties, presumably by their action as calcium buffers. The importance of calcium-binding proteins for nervous system function in an intact integrated system is, however, less clear. To investigate the physiological role of a major endogenous calcium-binding protein, calbindin D28k (calbindin)
in vivo
, we have generated calbindin null mutant mice by gene targeting. Surprisingly, calbindin deficiency does not affect general parameters of development and behavior or the structure of the nervous system at the light microscopic level. Null mutants are, however, severely impaired in tests of motor coordination, suggesting functional deficits in cerebellar pathways. Purkinje neurons, the only efferent of the cerebellar cortex, and inferior olive neurons, the source of the climbing fiber afferent, have previously been shown to express calbindin. Correlated with this unusual type of ataxia, confocal calcium imaging of Purkinje cells in cerebellar slices revealed marked changes of synaptically evoked postsynaptic calcium transients. Their fast, but not their slow, decay component had larger amplitudes in null mutant than in wild-type mice. We conclude that endogenous calbindin is of crucial importance for integrated nervous system function.
Benzo[
a
]pyrene diol epoxide-DNA cis adduct formation through a trans chlorohydrin intermediate
Authors: Meehan Thomas , Wolfe Alan R. , Negrete George R. , Song Qi
Published: July 2002

Alkylation of DNA by 7r,8t-dihydroxy,9t,10t-epoxy-7,8,9,10-tetrahydrobenzo[
a
]pyrene (
anti
-BPDE) forms mainly trans adducts (with respect to the C-9/10 positions). We recently described a halide-catalyzed pathway that preferentially generates cis adducts and now report that the trans chlorohydrin of
anti
-BPDE (
trans
-BPDCH) is an intermediate in the chloride-catalyzed reaction.
trans
-BPDCH was synthesized, and both it and
anti
-BPDE were reacted with deoxyadenosine as a model DNA nucleophile. The stereochemistry and yields of deoxyadenosine adducts were determined as a function of chloride concentration. In the absence of salt, the fraction of cis adducts obtained from
anti
-BPDE and
trans
-BPDCH are 0.33 and 0.67, respectively. Adding sodium chloride increases the fraction of cis adducts (and consequently decreases the fraction of trans adducts), with the midpoint of the increase for both substrates at approximately 35–40 mM chloride. The chloride-dependent curves for BPDE and BPDCH converge at 1 M chloride, where the fraction of cis adducts is 0.88. Chloride also increases the total yield of cis adducts with either substrate, whereas the yield of trans adducts from the chlorohydrin is not significantly changed. These results support a mechanism by which chloride ion undergoes nucleophilic addition to the benzylic C-10 position of
anti
-BPDE. This generates a trans halohydrin that alkylates DNA with inversion of configuration to form a cis adduct. This pathway may have biological significance because chlorohydrins could form in serum or in cells with relatively high intracellular concentrations of chloride.
Inhibition by uridine but not thymidine of p53-dependent intestinal apoptosis initiated by 5-fluorouracil: Evidence for the involvement of RNA perturbation
Authors: Pritchard D. Mark , Watson Alastair J. M. , Potten Christopher S. , Jackman Ann L. , Hickman John A.
Published: July 2002

The epithelia from the crypts of the intestine are exquisitely sensitive to metabolic perturbation and undergo cell death with the classical morphology of apoptosis. Administration of 40 mg/kg 5-fluorouracil (5-FU) to BDF-1 p53+/+ mice resulted in an increase in p53 protein at cell positions in the crypts that were also those subjected to an apoptotic cell death. In p53−/− mice apoptosis was almost completely absent, even after 24 hr. 5-FU is a pyrimidine antimetabolite cytotoxin with multiple mechanisms of action, including inhibition of thymidylate synthase (TS), which gives rise to DNA damage, and incorporation into RNA. The inhibition of TS can be increased by coadministration of folinic acid and can be abrogated by administration of thymidine. The incorporation of 5-FU into RNA is inhibited by administration of uridine. p53-Dependent cell death induced by 5-FU was only inhibited by administration of uridine. Uridine had no effect on the apoptosis initiated by 1 Gy of γ-radiation. Although thymidine abrogated apoptosis induced by the pure TS inhibitor Tomudex, it had no effect on 5-FU-induced apoptosis, and coadministration of folinic acid did not increase apoptosis. The data show that 5-FU-induced cell death of intestinal epithelial cells is p53-dependent and suggests that changes in RNA metabolism initiate events culminating in the expression of p53.
Inflammation and NOx-induced nitration: Assay for 3-nitrotyrosine by HPLC with electrochemical detection
Authors: Shigenaga Mark K. , Lee Ho H. , Blount Benjamin C. , Christen Stephan , Shigeno Eric T. , Yip Helena , Ames Bruce N.
Published: July 2002

The identification of15N-labeled 3-nitrotyrosine (NTyr) by gas chromatography/mass spectroscopy in protein hydrolyzates from activated RAW 264.7 macrophages incubated with15N-l-arginine confirms that nitric oxide synthase (NOS) is involved in the nitration of protein-bound tyrosine (Tyr). An assay is presented for NTyr that employs HPLC with tandem electrochemical and UV detection. The assay involves enzymatic hydrolysis of protein, acetylation, solvent extraction,O-deacetylation, and dithionite reduction to produce an analyte containingN-acetyl-3-aminotyrosine, an electrochemically active derivative of NTyr. We estimate the level of protein-bound NTyr in normal rat plasma to be ≈0–1 residues per 106Tyr with a detection limit of 0.5 per 107Tyr when ≥100 nmol of Tyr is analyzed and when precautions are taken to limit nitration artifacts. Zymosan-treated RAW 264.7 cells were shown to have an ≈6-fold higher level of protein-bound NTyr compared with control cells and cells treated withNG-monomethyl-l-arginine, an inhibitor of NOS. Intraperitoneal injection of F344 rats with zymosan led to a marked elevation in protein-bound NTyr to ≈13 residues per 106Tyr, an ≈40-fold elevation compared with plasma protein of untreated rats; cotreatment withNG-monomethyl-l-arginine inhibited the formation of NTyr in plasma protein from blood and peritoneal exudate by 69% and 53%, respectively. This assay offers a highly sensitive and quantitative approach for investigating the role of reactive byproducts of nitric oxide in the many pathological conditions and disease states associated with NOxexposure such as inflammation and smoking.
Development of a novel anti-HIV-1 agent from within: Effect of chimeric Vpr-containing protease cleavage site residues on virus replication
Authors: Serio D. , Rizvi T. A. , Cartas M. , Kalyanaraman V. S. , Weber I. T. , Koprowski H. , Srinivasan A.
Published: July 2002

Effective antiviral agents will be of great value in controlling virus replication and delaying the onset of HIV-1-related disease symptoms. Current therapy involves the use of antiviral agents that target the enzymatic functions of the virus, resulting in the emergence of resistant viruses to these agents, thus lowering their effectiveness. To overcome this problem, we have considered the idea of developing novel agents from within HIV-1 as inhibitors of virus replication. The specificity of the Vpr protein for the HIV-1 virus particle makes it an attractive molecule for the development of antiviral agents targeting the events associated with virus maturation. We have generated chimeric Vpr proteins containing HIV-1-specific sequences added to the C terminus of Vpr. These sequences correspond to nine cleavage sites of the Gag and Gag–Pol precursors of HIV-1. The chimeric Vpr constructs were introduced into HIV-1 proviral DNA to assess their effect on virus infectivity using single- and multiple-round replication assays. The virus particles generated exhibited a variable replication pattern depending on the protease cleavage site used as a fusion partner. Interestingly, the chimeric Vpr containing the cleavage sequences from the junction of p24 and p2, 24/2, completely abolished virus infectivity. These results show that chimeric proteins generated from within HIV-1 have the ability to suppress HIV-1 replication and make ideal agents for gene therapy or intracellular immunization to treat HIV-1 infection.
Familial multiple system tauopathy with presenile dementia: A disease with abundant neuronal and glial tau filaments
Authors: Spillantini Maria Grazia , Goedert Michel , Crowther R. Anthony , Murrell Jill R. , Farlow Martin R. , Ghetti Bernardino
Published: July 2002

Neurofibrillary lesions made of hyperphosphorylated microtubule-associated protein tau constitute not only one of the defining neuropathological features of Alzheimer disease but also are present in a number of other neurodegenerative diseases with dementia. Here we describe a novel autosomal dominant disease named familial “multiple system tauopathy with presenile dementia,” which is characterized by abundant fibrillary deposits of tau protein in both neurons and glial cells. There are no detectable deposits of β-amyloid. The tau deposits are in the form of twisted filaments that differ in diameter and periodicity from the paired helical filaments of Alzheimer disease. They are stained by both phosphorylation-independent and -dependent anti-tau antibodies. Moreover, tau immunoreactivity coexists with heparan sulfate in affected nerve and glial cells. Tau protein extracted from filaments of familial multiple system tauopathy with presenile dementia shows a minor 72-kDa band and two major bands of 64 and 68 kDa that contain mainly hyperphosphorylated four-repeat tau isoforms of 383 and 412 amino acids.
Self and viral peptides can initiate lysis by autologous natural killer cells
Authors: Mandelboim Ofer , Wilson S. Brian , Valés-Gómez Mar , Reyburn Hugh T. , Strominger Jack L.
Published: July 2002

Natural killer (NK) cells are inhibited by specific allotypes of class I major histocompatibility complex ligands recognized by polymorphic inhibitory receptors (e.g., NKIR1 and NKIR2). NK1- and NK2-specific clones recognize two groups of HLA-C allotypes that are distinguished by a dimorphism at residue 80 in the α1 helix (αLys-80 and αAsn-80, respectively). “Empty” HLA-Cw7 expressed in peptide transporter-deficient cells and HLA-Cw7 loaded with several peptides each functioned as inhibitory ligands for NK2 lines and clones. However, loading of HLA-Cw7 with two other peptides derived from glutamic acid decarboxylase or coxsackie virus (each of which has been associated with autoimmune diabetes mellitus) abrogated this inhibitory recognition. Both peptides contained Lys at P8 of the epitope. Substitution of P8 with Ala or two other basic amino acids, His and Arg, resulted in peptides that were inhibitory, as were peptides with P8 Val, Glu, or Asn. The manner in which a Lys at P8 might affect recognition is discussed, together with a hypothesis for a novel mechanism by which an autoimmune disease might be initiated.
Evolution of the primate lineage leading to modern humans: Phylogenetic and demographic inferences from DNA sequences
Authors: Takahata Naoyuki , Satta Yoko
Published: July 2002

To date major divergences that occurred in the primate lineage leading to modern humans and to infer a demographic parameter (effective population size) of the ancestral lineage that existed at each divergence, a maximum likelihood method was applied to autosomal DNA sequence data currently available for pairs of orthologous genes between the human and each of the chimpanzee, gorilla, Old World monkey (OWM), and New World monkey (NWM). A statistical test is carried out to support the assumption that silent substitutions have accumulated in a clock-like fashion over loci between primate taxa or even among sites within a locus. It is shown that the human ancestral lineage became distinct from the NWM 57.5 million years (Myr) ago, the OWM 31 Myr ago, the gorilla 8.0 Myr ago, and the chimpanzee 4.5 Myr ago, and that the effective population size at these divergences was generally much greater than that of modern humans. It is argued that the human ancestral lineage branched off from the NWM and OWM earlier than once thought and that significant demographic changes might have occurred at different evolutionary stages, particularly at the hominid stage.
Identification of an additional negative regulatory region for p53 sequence-specific DNA binding
Authors: Müller-Tiemann Beate F. , Halazonetis Thanos D. , Elting James J.
Published: July 2002

The DNA binding activity of p53 is crucial for its tumor suppressor function and is subject to tight regulation. Previous studies revealed that the inhibitory function of the p53 C terminus is implicated in the latent, low affinity sequence-specific DNA binding activity of p53 in the uninduced state. Sequence-specific DNA binding of p53 has been shown to be activated by several posttranslational modifications and interacting proteins that target predominantly the C terminus. Moreover, several authors have shown that synthetic peptides corresponding to p53 C-terminal sequences activate p53 sequence-specific DNA binding. In an effort to identify the interaction site of p53 with these activating peptides we assessed complex formation between p53 deletion constructs and C-terminal activating peptides by peptide affinity precipitation. This study revealed that two distal regions of the p53 molecule contribute synergistically to the interaction with activating C-terminal peptides: amino acids 80–93 and 364–393. The C-terminal residues 364–393 are already well characterized as having negative regulatory function. DNA binding analyses with these deletion constructs reveal a comparable negative regulatory activity for residues 80–93, defining this region as a previously unidentified negative regulatory domain of p53. Furthermore, synthetic peptides spanning this newly identified proline-rich negative regulatory region (residues 80–93) are able to activate p53 sequence-specific DNA binding
in vitro
. We suggest that both negative regulatory regions, residues 80–93 and 364–393, contribute cooperatively to the maintenance of the latent, low-affinity DNA binding conformation of p53.
Visualizing the dynamics of T cell activation: Intracellular adhesion molecule 1 migrates rapidly to the T cell/B cell interface and acts to sustain calcium levels
Authors: Wülfing Christoph , Sjaastad Michael D. , Davis Mark M.
Published: July 2002

T cell recognition typically involves both the engagement of a specific T cell receptor with a peptide/major histocompatibility complex (MHC) and a number of accessory interactions. One of the most important interactions is between the integrin lymphocyte function-associated antigen 1 (LFA-1) on the T cell and intracellular adhesion molecule 1 (ICAM-1) on an antigen-presenting cell. By using fluorescence video microscopy and an ICAM-1 fused to a green fluorescent protein, we find that the elevation of intracellular calcium in the T cell that is characteristic of activation is followed almost immediately by the rapid accumulation of ICAM-1 on a B cell at a tight interface between the two cells. This increased density of ICAM-1 correlates with the sustained elevation of intracellular calcium in the T cell, known to be critical for activation. The use of peptide/MHC complexes and ICAM-1 on a supported lipid bilayer to stimulate T cells also indicates a major role for ICAM-1/LFA-1 in T cell activation but, surprisingly, not for adhesion, as even in the absence of ICAM-1 the morphological changes and adhesive characteristics of an activated T cell are seen in this system. We suggest that T cell antigen receptor-mediated recognition of a very small number of MHC/peptide complexes could trigger LFA-1/ICAM-1 clustering and avidity regulation, thus amplifying and stabilizing the production of second messengers.
p21
WAF1
is required for butyrate-mediated growth inhibition of human colon cancer cells
Authors: Archer Sonia Y. , Meng Shufen , Shei Amy , Hodin Richard A.
Published: July 2002

A diet high in fiber is associated with a decreased incidence and growth of colon cancers. Butyrate, a four-carbon short-chain fatty acid product of fiber fermentation within the colon, appears to mediate these salutary effects. We sought to determine the molecular mechanism by which butyrate mediates growth inhibition of colonic cancer cells and thereby to elucidate the molecular link between a high-fiber diet and the arrest of colon carcinogenesis. We show that concomitant with growth arrest, butyrate induces p21 mRNA expression in an immediate-early fashion, through transactivation of a promoter cis-element(s) located within 1.4 kb of the transcriptional start site, independent of p53 binding. Studies using the specific histone hyperacetylating agent, trichostatin A, and histone deacetylase 1 indicate that growth arrest and p21 induction occur through a mechanism involving histone hyperacetylation. We show the critical importance of p21 in butyrate-mediated growth arrest by first confirming that stable overexpression of the p21 gene is able to cause growth arrest in the human colon carcinoma cell line, HT-29. Furthermore, using p21-deleted HCT116 human colon carcinoma cells, we provide convincing evidence that p21 is required for growth arrest to occur in response to histone hyperacetylation, but not for serum starvation nor postconfluent growth. Thus, p21 appears to be a critical effector of butyrate-induced growth arrest in colonic cancer cells, and may be an important molecular link between a high-fiber diet and the prevention of colon carcinogenesis.
Late-onset minor and major depression: early evidence for common neuroanatomical substrates detected by using MRI
Authors: Kumar Anand , Jin Zhisong , Bilker Warren , Udupa Jayaram , Gottlieb Gary
Published: July 2002

The purpose of our study was to examine the neuroanatomical correlates of late-onset minor and major depression and to compare them with similar measures obtained from nondepressed controls. Our study groups were comprised of 18 patients with late-onset minor depression, 35 patients diagnosed with late-onset major depression, and 30 nondepressed controls. All subjects were scanned by using a 1.5-tesla MRI scanner. Absolute whole brain volume and normalized measures of prefrontal and temporal lobe volumes were obtained and used for comparison among groups. Our findings indicate that patients with minor depression present with specific neuroanatomical abnormalities that are comparable with the major depression group but significantly different from the controls. Normalized prefrontal lobe volumes show a significant linear trend with severity of depression, with volumes decreasing with illness severity. Whole brain volumes did not differ significantly among groups. These findings have broad implications for the biology of late-life depression and suggest that there may be common neurobiological substrates that underlie all clinically significant forms of late-onset mood disturbances.
Antisense oligonucleotides against α
1E
reduce R-type calcium currents in cerebellar granule cells
Authors: Piedras-Rentería Erika S. , Tsien Richard W.
Published: July 2002

Many neurons of the central nervous system display multiple high voltage-activated Ca
2+
currents, pharmacologically classified as L-, N-, P-, Q-, and R-type. Of these current types, the R-type is the least understood. The leading candidate for the molecular correlate of R-type currents in cerebellar granule cells is the α
1E
subunit, which yields Ca
2+
currents very similar to the R-type when expressed in heterologous systems. As a complementary approach, we tested whether antisense oligonucleotides against α
1E
could decrease the expression of R-type current in rat cerebellar granule neurons in culture. Cells were supplemented with either antisense or sense oligonucleotides and whole-cell patch clamp recordings were obtained after 6–8 days
in vitro
. Incubation with α
1E
antisense oligonucleotide caused a 52.5% decrease in the peak R-type current density, from −10 ± 0.6 picoamperes/picofarad (pA/pF) (
n
= 6) in the untreated controls to −4.8 ± 0.8 pA/pF (
n
= 11) (
P
< 0.01). In contrast, no significant changes in the current expression were seen in sense oligonucleotide-treated cells (−11.3 ± 3.2 pA/pF). The specificity of the α
1E
antisense oligonucleotides was supported by the lack of change in estimates of the P/Q current amplitude. Furthermore, antisense and sense oligonucleotides against α
1A
did not affect R-type current expression (−11.5 ± 1.7 and −11.7 ± 1.7 pA/pF, respectively), whereas the α
1A
antisense oligonucleotide significantly reduced whole cell currents under conditions in which P/Q current is dominant. Our results support the hypothesis that members of the E class of α
1
subunits support the high voltage-activated R-type current in cerebellar granule cells.
Quasi-axially symmetric stellarators
Authors: Garabedian Paul R.
Published: July 2002

Confinement of a plasma for controlled thermonuclear fusion is studied numerically. Toroidal equilibria are considered, with an emphasis on the Modular Helias-like Heliac 2 (MHH2), which is a stellarator of low aspect ratio with just two field periods surrounded by 16 modular coils. The geometry is fully three-dimensional, but there is an axial symmetry of the magnetic structure that is calculated to give confinement competitive with that in circular tokamaks. Additional vertical and toroidal field coils, together with a current drive, provide the flexibility and the control of rotational transform necessary for a successful experiment. An MHH3 device with three field periods and comparable quasi-axial symmetry is presented, too, and because of reversed shear, its physical properties may be better. Variational analysis of equilibrium and stability is shown to give a more reliable prediction of performance for these stellarators than linearized or local theories that suffer from a failure of differentiability and convergence.
Transport of uncharged organic solutes in
Xenopus
oocytes expressing red cell anion exchangers (AE1s)
Authors: Fiévet Bruno , Perset Frédéric , Gabillat Nicole , Guizouarn Hélène , Borgese Franck , Ripoche Pierre , Motais René
Published: July 2002

When expressed in
Xenopus
oocytes, the trout red cell anion exchanger tAE1, but not the mouse exchanger mAE1, elicited a transport of electroneutral solutes (sorbitol, urea) in addition to the expected anion exchange activity. Chimeras constructed from mAE1 and tAE1 allowed us to identify the tAE1 domains involved in the induction of these transports. Expression of tAE1 (but not mAE1) is known to generate an anion conductance associated with a taurine transport. The present data provide evidence that (
i
) the capacity of tAE1 and tAE1 chimeras to generate urea and sorbitol permeability also was associated with an anion conductance; (
ii
) the same inhibitors affected both the permeability of solutes and anion conductance; and (
iii
) no measurable water transport was associated with the tAE1-dependent conductance. These results support the view that fish red blood cells, to achieve cell volume regulation in response to hypotonic swelling, activate a tAE1-associated anion channel that can mediate the passive transport of taurine and electroneutral solutes.
Structural analysis of cloned plasma membrane proteins by freeze-fracture electron microscopy
Authors: Eskandari Sepehr , Wright Ernest M. , Kreman Mike , Starace Dorine M. , Zampighi Guido A.
Published: July 2002

We have used freeze-fracture electron microscopy to examine the oligomeric structure and molecular asymmetry of integral plasma membrane proteins. Recombinant plasma membrane proteins were functionally expressed in
Xenopus laevis
oocytes, and the dimensions of their freeze-fracture particles were analyzed. To characterize the freeze-fracture particles, we compared the particle cross-sectional area of proteins with α-helical transmembrane domains (opsin, aquaporin 1, and a connexin) with their area obtained from existing maps calculated from two-dimensional crystals. We show that the cross-sectional area of the freeze-fracture particles corresponds to the area of the transmembrane domain of the protein, and that the protein cross-sectional area varies linearly with the number membrane-spanning helices. On average, each helix occupies 1.40 ± 0.03 nm
2
. By using this information, we examined members from three classes of plasma membrane proteins: two ion channels, the cystic fibrosis transmembrane conductance regulator and connexin 50 hemi-channel; a water channel, the major intrinsic protein (the aquaporin 0); and a cotransporter, the Na
+
/glucose cotransporter. Our results suggest that the cystic fibrosis transmembrane conductance regulator is a dimer containing 25 ± 2 transmembrane helices, connexin 50 is a hexamer containing 24 ± 3 helices, the major intrinsic protein is a tetramer containing 24 ± 3 helices, and the Na
+
/glucose cotransporter is an asymmetrical monomer containing 15 ± 2 helices.
The role of the ClpA chaperone in proteolysis by ClpAP
Authors: Hoskins Joel R. , Pak Marie , Maurizi Michael R. , Wickner Sue
Published: July 2002

ClpA, a member of the Clp/Hsp100 family of ATPases, is a molecular chaperone and, in combination with a proteolytic component ClpP, participates in ATP-dependent proteolysis. We investigated the role of ClpA in protein degradation by ClpAP by dissociating the reaction into several discrete steps. In the assembly step, ClpA–ClpP–substrate complexes assemble either by ClpA–substrate complexes interacting with ClpP or by ClpA–ClpP complexes interacting with substrate; ClpP in the absence of ClpA is unable to bind substrates. Assembly requires ATP binding but not hydrolysis. We discovered that ClpA translocates substrates from their binding sites on ClpA to ClpP. The translocation step specifically requires ATP; nonhydrolyzable ATP analogs are ineffective. Only proteins that are degraded by ClpAP are translocated. Characterization of the degradation step showed that substrates can be degraded in a single round of ClpA–ClpP–substrate binding followed by ATP hydrolysis. The products generated are indistinguishable from steady-state products. Taken together, our results suggest that ClpA, through its interaction with both the substrate and ClpP, acts as a gatekeeper, actively translocating specific substrates into the proteolytic chamber of ClpP where degradation occurs. As multicomponent ATP-dependent proteases are widespread in nature and share structural similarities, these findings may provide a general mechanism for regulation of substrate import into the proteolytic chamber.
Induction of microsatellite instability by oxidative DNA damage
Authors: Jackson Aimee L. , Chen Ru , Loeb Lawrence A.
Published: July 2002

Instability of repetitive sequences, both in intronic sequences and within coding regions, has been demonstrated to be a hallmark of genomic instability in human cancer. Understanding how these mutational events arise may provide an opportunity for prevention or early intervention in cancer development. To study the source of this instability, we have identified a region of the β-lactamase gene that is tolerant to the insertion of fragments of exogenous DNA as large as 1,614 bp with minimal loss of enzyme activity, as determined by antibiotic resistance. Fragments inserted out-of-frame render
Escherichia coli
sensitive to antibiotic, and compensatory frameshift mutations that restore the reading frame of β-lactamase can be selected on the basis of antibiotic resistance. We have utilized this site to insert a synthetic microsatellite sequence within the β-lactamase gene and selected for mutations yielding frameshifts. This assay provides for detection of one frameshift mutation in a background of 10
6
wild-type sequences. Mismatch repair deficiency increased the observed frameshift frequency ≈300-fold. Exposure of plasmid containing microsatellite sequences to hydrogen peroxide resulted in frameshift mutations that were localized exclusively to the microsatellite sequences, whereas DNA damage by UV or
N
-methyl-
N
′-nitro-
N
-nitrosoguanidine did not result in enhanced mutagenesis. We postulate that in tumor cells, endogenous production of oxygen free radicals may be a major factor in promoting instability of microsatellite sequences. This β-lactamase assay may provide a sensitive methodology for the detection and quantitation of mutations associated with the development of cancer.
Endothelin 3 selectively promotes survival and proliferation of neural crest-derived glial and melanocytic precursorsin vitro
Authors: Lahav Ronit , Dupin Elisabeth , Lecoin Laure , Glavieux Corinne , Champeval Delphine , Ziller Catherine , Le Douarin Nicole M.
Published: July 2002

Genetic data in the mouse have shown that endothelin 3 (ET3) and its receptor B (ETRB) are essential for the development of two neural crest (NC) derivatives, the melanocytes and the enteric nervous system. We report here the effects of ET3in vitroon the differentiation of quail trunk NC cells (NCC) in mass and clonal cultures. Treatment with ET3 is highly mitogenic to the undifferentiated NCC population, which leads to expansion of the population of cells in the melanocytic, and to a lesser extent, the glial lineages. The effect of ET3 on these two NC derivatives was confirmed by the quantitative analysis of clones derived from individual NCC subjected to ET3: we found a large increase in the survival and proliferation of unipotent and bipotent precursors for glial cells and melanocytes, with no significant effect on multipotent cells generating neurons. ET3 first stimulates expression of both ETRB and ETRB2 by cultured NCC. Then, under prolonged exposure to ET3, ETRB expression decreases and switches toward an ETRB2-positive melanogenic cell population. We therefore propose that the presentin vitroexperiments (long-lasting exposure to a high concentration of ET3) mimic the environment encountered by NCCin vivowhen they migrate to the skin under the ectoderm that expresses ET3.
Tarsier-like locomotor specializations in the Oligocene primateAfrotarsius
Authors: Rasmussen D. Tab , Conroy Glenn C. , Simons Elwyn L.
Published: July 2002

Tarsiers and extinct tarsier-like primates have played a central role in views of primate phylogeny and evolution for more than a century. Because of the importance of tarsiers in so many primatological problems, there has been particular interest in questions about the origin of tarsier specializations and the biogeography of early tarsioid radiations. We report on a new fossil of rareAfrotarsiusthat shows near identity to modernTarsiusin unique specializations of the leg, which provides information about the locomotor behavior and clarifies the phylogenetic position of this previously controversial primate. These specializations constitute evidence thatAfrotarsiusis a tarsiid, closely related to extantTarsius; hence, it is now excluded from being a generalized sister taxon to Anthropoidea.
HNS, a nuclear-cytoplasmic shuttling sequence in HuR
Authors: Fan Xinhao Cynthia , Steitz Joan A.
Published: July 2002

Proteins are transported into and out of the cell nucleus via specific signals. The two best-studied nuclear transport processes are mediated either by classical nuclear localization signals or nuclear export signals. There also are shuttling sequences that direct the bidirectional transport of RNA-binding proteins. Two examples are the M9 sequence in heterogeneous nuclear ribonucleoprotein A1 and the heterogeneous nuclear ribonucleoprotein K shuttling domain (KNS) sequence in heterogeneous nuclear ribonucleoprotein K, both of which appear to contribute importantly to the export of mRNA to the cytoplasm. HuR is an RNA-binding protein that can stabilize labile mRNAs containing AU-rich elements in their 3′ untranslated regions and has been shown to shuttle between the nucleus and cytoplasm (18, 19). We have identified in HuR a shuttling sequence that also possess transcription-dependent nuclear localization signal activity. We propose that HuR first may bind AU-rich element-containing mRNAs in the nucleus and then escort them through the nuclear pore, providing protection during and after export to the cytoplasmic compartment.
A subclass of prefrontal γ-aminobutyric acid axon terminals are selectively altered in schizophrenia
Authors: Woo Tsung-Ung , Whitehead Richard E. , Melchitzky Darlene S. , Lewis David A.
Published: July 2002

In the primate cerebral cortex, morphologically and functionally diverse classes of local circuit neurons containing the inhibitory neurotransmitter γ-aminobutyric acid (GABA) differentially regulate the activity of pyramidal cells, the principal type of excitatory output neurons. In schizophrenia, GABA neurotransmission in the prefrontal cortex (PFC) appears to be disturbed but whether specific populations of GABA neurons are affected is not known. The chandelier class of GABA neurons are of particular interest because their axon terminals, which form distinctive arrays termed “cartridges,” provide inhibitory input exclusively to the axon initial segment of pyramidal cells. Thus, chandelier cells are positioned to powerfully regulate the excitatory output of pyramidal neurons and, consequently, to substantially affect the patterns of neuronal activity within the PFC. In this study, an antibody directed against the GABA membrane transporter GAT-1 was used to label GABA axon terminals in postmortem human brains. The relative density of GAT-1-immunoreactive axon cartridges furnished by chandelier neurons was decreased by 40% in the PFC of schizophrenic subjects compared with matched groups of normal control and nonschizophrenic psychiatric subjects. In contrast, markers of the axon terminals of other populations of GABA neurons were not altered in the schizophrenic subjects. Furthermore, the density of GAT-1-immunoreactive axon cartridges was not altered in psychiatric subjects who had been treated with antipsychotic medications. The changes in GAT-1-immunoreactive axon cartridges of chandelier neurons in schizophrenia are likely to reflect altered information processing within the PFC and in its output connections to other brain regions and could contribute to the cognitive impairments seen in this disorder.