bluuebunny/crossref_metadata_2025 huggingface dataset:
Mitogen-activated protein kinase-mediated Fas apoptotic signaling pathway
Authors: Goillot Evelyne , Raingeaud Joel , Ranger Ann , Tepper Robert I. , Davis Roger. J. , Harlow Ed , Sanchez Irma
Published: July 2002

Ligation of the cell surface receptor Fas/APO-1 (CD95) by its specific ligand or by anti-Fas antibodies rapidly induces apoptosis in susceptible cells. To characterize the molecular events involved in Fas-induced apoptosis, we examined the contribution of two subgroups of the mitogen-activated protein (MAP) kinase family, the Jun kinases or stress-activated protein kinases (JNKs/SAPKs) and the extracellular signal-regulated kinases (ERKs), in a Fas-sensitive neuroblastoma cell line. Here we show that both JNK and ERK protein kinases were activated upon Fas crosslinking through a Ras-dependent mechanism. Interference with either the JNK or ERK pathway by ectopic expression of dominant-interfering mutant proteins blocked Fas-mediated apoptosis. ERK activation was transient and associated with induced expression of the Fas receptor. In contrast, JNK activation was sustained and correlated with the onset of apoptosis. These data indicate that the ERK and the JNK groups of MAP kinases cooperate in the induction of cell death by Fas. Inhibition of Fas killing by an interleukin 1β-converting enzyme (ICE)-like protease inhibitor peptide did not modify Fas-induced JNK activation upon Fas ligation. In contrast, changes in Bcl-2 level due to expression of sense and antisense vectors influenced the sensitivity to Fas killing and Fas-induced JNK activation.
Increased substance P responses in dorsal root ganglia and intestinal macrophages duringClostridium difficiletoxin A enteritis in rats
Authors: Castagliuolo Ignazio , Keates Andrew C. , Qiu Bosheng , Kelly Ciaran P. , Nikulasson Sigfus , Leeman Susan E. , Pothoulakis Charalabos
Published: July 2002

Previously we reported that pretreatment of rats with the substance P (SP) antagonist CP-96,345 inhibits the enterotoxic responses following administration of toxin A fromClostridium difficileinto ileal loops, indicating that SP participates in the intestinal responses to this toxin. We now report that injection of toxin A into rat ileum causes a rapid increase in SP content in lumbar dorsal root ganglia (DRG) and mucosal scrapings 30–60 min after toxin A administration. Toxin A-mediated fluid secretion, mannitol permeability, and ileal histologic damage is significantly increased only after 2 hr. Toxin A also causes an increase in the abundance of SP mRNA in lumbar DRG and ileal mucosa as measured by reverse transcription–PCR. Lamina propria macrophages (LPMs) obtained from toxin A-injected loops release greater amounts of tumor necrosis factor α (TNFα) and SP as compared with LPMs isolated from buffer-injected loops (P< 0.01). Pretreatment of rats with the SP antagonist CP-96,345 inhibits toxin A-mediated TNFα release from isolated LPMs, whereas an inactive enantiomer (CP-96,344) of the SP antagonist has no effect. LPMs obtained from toxin A-injected ileal loops incubatedin vitrowith SP (10−8to 10−9M) show enhanced TNFα secretion, whereas LPMs isolated from buffer-injected loops do not respond to SP. In addition, LPMs obtained from toxin A-injected ileal loops incubatedin vitrowith CP-96,345 showed a diminished TNFα release. Our results indicate that activated LPMs secrete SP during toxin A enteritis that can lead to secretion of cytokines, suggesting an autocrine/paracrine regulation of cytokine secretion by SP from LPMs during intestinal inflammation.
The SbcCD nuclease of
Escherichia coli
is a structural maintenance of chromosomes (SMC) family protein that cleaves hairpin DNA
Authors: Connelly John C. , Kirkham Lucy A. , Leach David R. F.
Published: July 2002

Hairpin structures can inhibit DNA replication and are intermediates in certain recombination reactions. We have shown that the purified SbcCD protein of
Escherichia coli
cleaves a DNA hairpin. This cleavage does not require the presence of a free (3′ or 5′) DNA end and generates products with 3′-hydroxyl and 5′-phosphate termini. Electron microscopy of SbcCD has revealed the “head-rod-tail” structure predicted for the SMC (structural maintenance of chromosomes) family of proteins, of which SbcC is a member. This work provides evidence consistent with the proposal that SbcCD cleaves hairpin structures that halt the progress of the replication fork, allowing homologous recombination to restore DNA replication.
Stress signaling through Ca
2+
/calmodulin-dependent protein phosphatase calcineurin mediates salt adaptation in plants
Authors: Pardo José M. , Reddy Muppala P. , Yang Shuli , Maggio Albino , Huh Gyung-Hye , Matsumoto Tracie , Coca Maria A. , Paino-D’Urzo Matilde , Koiwa Hisashi , Yun Dae-Jin , Watad Abed A. , Bressan Ray A. , Hasegawa Paul M.
Published: July 2002

Calcineurin (CaN) is a Ca
2+
- and calmodulin-dependent protein phosphatase (PP2B) that, in yeast, is an integral intermediate of a salt-stress signal transduction pathway that effects NaCl tolerance through the regulation of Na
+
influx and efflux. A truncated form of the catalytic subunit and the regulatory subunit of yeast CaN were coexpressed in transgenic tobacco plants to reconstitute a constitutively activated phosphatase
in vivo
. Several different transgenic lines that expressed activated CaN also exhibited substantial NaCl tolerance, and this trait was linked to the genetic inheritance of the CaN transgenes. Enhanced capacity of plants expressing CaN to survive NaCl shock was similar when evaluation was conducted on seedlings in tissue culture raft vessels or plants in hydroponic culture that were transpiring actively. Root growth was less perturbed than shoot growth by NaCl in plants expressing CaN. Also, NaCl stress survival of control shoots was enhanced substantially when grafted onto roots of plants expressing CaN, further implicating a significant function of the phosphatase in the preservation of root integrity during salt shock. Together, these results indicate that in plants, like in yeast, a Ca
2+
- and calmodulin-dependent CaN signal pathway regulates determinants of salt tolerance required for stress adaptation. Furthermore, modulation of this pathway by expression of an activated regulatory intermediate substantially enhanced salt tolerance.
Unexpected frameshifts from gene to expressed protein in a phage-displayed peptide library
Authors: Cárcamo Juan , Ravera Mark W. , Brissette Renée , Dedova Olga , Beasley James R. , Alam-Moghé Ameena , Wan Changhong , Blume Arthur , Mandecki Wlodek
Published: July 2002

A library of long peptides displayed on the pIII protein of filamentous phage was used in biopanning experiments against several protein targets. We find that a large percentage of phage clones that bind specifically to a target contain peptide-encoding genes that do not have an ORF. Instead, the reading frame is either interrupted by one or more nonsuppressed stop codons, or a post-transcriptional frameshift is needed to account for the expression of the minor phage coat protein pIII. The percentage of frameshifted clones varies depending on the target. It can be as high as 90% for clones specific for soluble forms of certain cytokine receptors. Conversely, biopanning against four mAbs did not yield any frameshifted clones. Our studies focused on one clone that binds specifically to rat growth hormone binding protein (GHBP) yet does not have an ORF. A secondary peptide library containing random mutations of this sequence was constructed and panned against GHBP to optimize and correct the reading frame. In the last round (round two) of panning with this library, none of the phage clones that bound to GHBP had an ORF. However, careful analysis of these clones allowed us to design a synthetic peptide capable of binding to GHBP. The results of this study indicate that ORFs are not required to obtain gene expression of the minor coat protein of filamentous phage and suggest that some ORF
−
clones may have a selective advantage over the clones having ORFs.
Transgenic mice for interleukin 3 develop motor neuron degeneration associated with autoimmune reaction against spinal cord motor neurons
Authors: Chavany Christine , Vicario-Abejón Carlos , Miller Georgina , Jendoubi Moncef
Published: July 2002

Interleukin 3 (IL-3) stimulates the proliferation and differentiation of various haematopoietic progenitor cells. Recently, IL-3 and other cytokines were reported to exert a neurotrophic activity and to be associated with neurological disorders, suggesting their complex role in the central nervous system. We now show that overexpression of IL-3 in transgenic mice causes a motor neuron disease with several features of amyotrophic lateral sclerosis and progressive muscular atrophy. These animals exhibit hind limb paralysis at 7 months of age, associated with dendritic and axonal degeneration, loss of motor neurons in the spinal cord, and autoimmune reaction against these cells. We examined the effect of IL-3 on embryonic motor neurons survival in mixed spinal cord cultures. Our results suggest that motor neuronal degeneration is not directly triggered by the high level of expression of IL-3.
RNA folding causes secondary structure rearrangement
Authors: Wu Ming , Tinoco Ignacio
Published: July 2002

The secondary structure of the P5abc subdomain (a 56-nt RNA) of the
Tetrahymena thermophila
group I intron ribozyme has been determined by NMR. Its base pairing in aqueous solution in the absence of magnesium ions is significantly different from the RNA in a crystal but is consistent with thermodynamic predictions. On addition of magnesium ions, the RNA folds into a tertiary structure with greatly changed base pairing consistent with the crystal structure: three Watson–Crick base pairs, three G⋅U base pairs, and an extra-stable tetraloop are lost. The common assumption that RNA folds by first forming secondary structure and then forming tertiary interactions from the unpaired bases is not always correct.
HIV-1 Tat binds TAFII250 and represses TAFII250-dependent transcription of major histocompatibility class I genes
Authors: Weissman Jocelyn D. , Brown Julie A. , Howcroft T. Kevin , Hwang Jae , Chawla Ashish , Roche Paul A. , Schiltz Louis , Nakatani Yoshihiro , Singer Dinah S.
Published: July 2002

HIV Tat, a transactivator of viral transcription, represses transcription of major histocompatibility (MHC) class I genes. Repression depends exclusively on the C-terminal domain of Tat, although the mechanism of this repression has not been known. We now show that repression results from the interaction of Tat with the TAFII250 component of the general transcription factor, TFIID. The C-terminal domain of Tat binds to a site on TAFII250 that overlaps the histone acetyl transferase domain, inhibiting TAFII250 histone acetyl transferase activity. Furthermore, promoters repressed by Tat, including the MHC class I promoter, are dependent on TAFII250 whereas those that are not repressed by Tat, such as SV40 and MuLV promoters, are independent of functional TAFII250. Thus, Tat repression of MHC class I transcription would be one mechanism by which HIV avoids immune surveillance.
From
ab initio
quantum mechanics to molecular neurobiology: A cation–π binding site in the nicotinic receptor
Authors: Zhong Wenge , Gallivan Justin P. , Zhang Yinong , Li Lintong , Lester Henry A. , Dougherty Dennis A.
Published: July 2002

The nicotinic acetylcholine receptor is the prototype ligand-gated ion channel. A number of aromatic amino acids have been identified as contributing to the agonist binding site, suggesting that cation–π interactions may be involved in binding the quaternary ammonium group of the agonist, acetylcholine. Here we show a compelling correlation between: (
i
)
ab initio
quantum mechanical predictions of cation–π binding abilities and (
ii
) EC
50
values for acetylcholine at the receptor for a series of tryptophan derivatives that were incorporated into the receptor by using the
in vivo
nonsense-suppression method for unnatural amino acid incorporation. Such a correlation is seen at one, and only one, of the aromatic residues—tryptophan-149 of the α subunit. This finding indicates that, on binding, the cationic, quaternary ammonium group of acetylcholine makes van der Waals contact with the indole side chain of α tryptophan-149, providing the most precise structural information to date on this receptor. Consistent with this model, a tethered quaternary ammonium group emanating from position α149 produces a constitutively active receptor.
A gene cluster for macrolide antibiotic biosynthesis in
Streptomyces venezuelae
: Architecture of metabolic diversity
Authors: Xue Yongquan , Zhao Lishan , Liu Hung-wen , Sherman David H.
Published: July 2002

In a survey of microbial systems capable of generating unusual metabolite structural variability,
Streptomyces venezuelae
ATCC 15439 is notable in its ability to produce two distinct groups of macrolide antibiotics. Methymycin and neomethymycin are derived from the 12-membered ring macrolactone 10-deoxymethynolide, whereas narbomycin and pikromycin are derived from the 14-membered ring macrolactone, narbonolide. This report describes the cloning and characterization of the biosynthetic gene cluster for these antibiotics. Central to the cluster is a polyketide synthase locus (
pikA
) that encodes a six-module system comprised of four multifunctional proteins, in addition to a type II thioesterase (TEII). Immediately downstream is a set of genes for desosamine biosynthesis (
des
) and macrolide ring hydroxylation. The study suggests that Pik TEII plays a role in forming a metabolic branch through which polyketides of different chain length are generated, and the glycosyl transferase (encoded by
desVII
) has the ability to catalyze glycosylation of both the 12- and 14-membered ring macrolactones. Moreover, the
pikC
-encoded P450 hydroxylase provides yet another layer of structural variability by introducing regiochemical diversity into the macrolide ring systems. The data support the notion that the architecture of the
pik
gene cluster as well as the unusual substrate specificity of particular enzymes contributes to its ability to generate four macrolide antibiotics.
Complete sequencing of the
Fugu
WAGR region from WT1 to PAX6: Dramatic compaction and conservation of synteny with human chromosome 11p13
Authors: Miles Colin , Elgar Greg , Coles Edward , Kleinjan Dirk-Jan , van Heyningen Veronica , Hastie Nicholas
Published: July 2002

The pufferfish
Fugu rubripes
has a genome ≈7.5 times smaller than that of mammals but with a similar number of genes. Although conserved synteny has been demonstrated between pufferfish and mammals across some regions of the genome, there is some controversy as to what extent
Fugu
will be a useful model for the human genome, e.g., [Gilley, J., Armes, N. & Fried, M. (1997)
Nature (London)
385, 305–306]. We report extensive conservation of synteny between a 1.5-Mb region of human chromosome 11 and <100 kb of the
Fugu
genome in three overlapping cosmids. Our findings support the idea that the majority of DNA in the region of human chromosome 11p13 is intergenic. Comparative analysis of three unrelated genes with quite different roles,
WT1
,
RCN1
, and
PAX6
, has revealed differences in their structural evolution. Whereas the human
WT1
gene can generate 16 protein isoforms via a combination of alternative splicing, RNA editing, and alternative start site usage, our data predict that
Fugu WT1
is capable of generating only two isoforms. This raises the question of the extent to which the evolution of
WT1
isoforms is related to the evolution of the mammalian genitourinary system. In addition, this region of the
Fugu
genome shows a much greater overall compaction than usual but with significant noncoding homology observed at the
PAX6
locus, implying that comparative genomics has identified regulatory elements associated with this gene.
The reactive site loop of the serpin SCCA1 is essential for cysteine proteinase inhibition
Authors: Schick Charles , Brömme Dieter , Bartuski Allison J. , Uemura Yoshiki , Schechter Norman M. , Silverman Gary A.
Published: July 2002

The high-molecular-weight serine proteinase inhibitors (serpins) are restricted, generally, to inhibiting proteinases of the serine mechanistic class. However, the viral serpin, cytokine response modifier A, and the human serpins, antichymotrypsin and squamous cell carcinoma antigen 1 (SCCA1), inhibit different members of the cysteine proteinase class. Although serpins employ a mobile reactive site loop (RSL) to bait and trap their target serine proteinases, the mechanism by which they inactivate cysteine proteinases is unknown. Our previous studies suggest that SCCA1 inhibits papain-like cysteine proteinases in a manner similar to that observed for serpin–serine proteinase interactions. However, we could not preclude the possibility of an inhibitory mechanism that did not require the serpin RSL. To test this possibility, we employed site-directed mutagenesis to alter the different residues within the RSL. Mutations to either the hinge or the variable region of the RSL abolished inhibitory activity. Moreover, RSL swaps between SCCA1 and the nearly identical serpin, SCCA2 (an inhibitor of chymotrypsin-like serine proteinases), reversed their target specificities. Thus, there were no unique motifs within the framework of SCCA1 that independently accounted for cysteine proteinase inhibitory activity. Collectively, these data suggested that the sequence and mobility of the RSL of SCCA1 are essential for cysteine proteinase inhibition and that serpins are likely to utilize a common RSL-dependent mechanism to inhibit both serine and cysteine proteinases.
Dynamics of fluorescence fluctuations in green fluorescent protein observed by fluorescence correlation spectroscopy
Authors: Haupts Ulrich , Maiti Sudipta , Schwille Petra , Webb Watt W.
Published: July 2002

We have investigated the pH dependence of the dynamics of conformational fluctuations of green fluorescent protein mutants EGFP (F64L/S65T) and GFP-S65T in small ensembles of molecules in solution by using fluorescence correlation spectroscopy (FCS). FCS utilizes time-resolved measurements of fluctuations in the molecular fluorescence emission for determination of the intrinsic dynamics and thermodynamics of all processes that affect the fluorescence. Fluorescence excitation of a bulk solution of EGFP decreases to zero at low pH (pK
a
= 5.8) paralleled by a decrease of the absorption at 488 nm and an increase at 400 nm. Protonation of the hydroxyl group of Tyr-66, which is part of the chromophore, induces these changes. When FCS is used the fluctuations in the protonation state of the chromophore are time resolved. The autocorrelation function of fluorescence emission shows contributions from two chemical relaxation processes as well as diffusional concentration fluctuations. The time constant of the fast, pH-dependent chemical process decreases with pH from 300 μs at pH 7 to 45 μs at pH 5, while the time-average fraction of molecules in a nonfluorescent state increases to 80% in the same range. A second, pH-independent, process with a time constant of 340 μs and an associated fraction of 13% nonfluorescent molecules is observed between pH 8 and 11, possibly representing an internal proton transfer process and associated conformational rearrangements. The FCS data provide direct measures of the dynamics and the equilibrium properties of the protonation processes. Thus FCS is a convenient, intrinsically calibrated method for pH measurements in subfemtoliter volumes with nanomolar concentrations of EGFP.
Evidence for a voltage-dependent enhancement of neurotransmitter release mediated via the synaptic protein interaction site of N-type Ca2+channels
Authors: Mochida Sumiko , Yokoyama Charles T. , Kim D. Kyle , Itoh Kanako , Catterall William A.
Published: July 2002

Secretion of neurotransmitters is initiated by voltage-gated calcium influx through presynaptic, voltage-gated N-type calcium channels. These channels interact with the SNARE proteins, which are core components of the exocytosis process, via the synaptic protein interaction (synprint) site in the intracellular loop connecting domains II and III of their α1Bsubunit. Interruption of this interaction by competing synprint peptides inhibits fast, synchronous transmitter release. Here we identify a voltage-dependent, but calcium-independent, enhancement of transmitter release that is elicited by trains of action potentials in the presence of a hyperosmotic extracellular concentration of sucrose. This enhancement of transmitter release requires interaction of SNARE proteins with the synprint site. Our results provide evidence for a voltage-dependent signal that is transmitted by protein–protein interactions from the N-type calcium channel to the SNARE proteins and enhances neurotransmitter release by altering SNARE protein function.
Identification and characterization of a retinoid-induced class II tumor suppressor/growth regulatory gene
Authors: DiSepio Daniel , Ghosn Corine , Eckert Richard L. , Deucher Anne , Robinson Nancy , Duvic Madeleine , Chandraratna Roshantha A. S. , Nagpal Sunil
Published: July 2002

Retinoids, synthetic and natural analogs of retinoic acid, exhibit potent growth inhibitory and cell differentiation activities that account for their beneficial effects in treating hyperproliferative diseases such as psoriasis, actinic keratosis, and certain neoplasias. Tazarotene is a synthetic retinoid that is used in the clinic for the treatment of psoriasis. To better understand the mechanism of retinoid action in the treatment of hyperproliferative diseases, we used a long-range differential display–PCR to isolate retinoid-responsive genes from primary human keratinocytes. We have identified a cDNA,tazarotene-induced gene 3(TIG3;Retinoic Acid Receptor Responder 3) showing significant homology to the class II tumor suppressor gene,H-rev107. Tazarotene treatment increasesTIG3expression in primary human keratinocytes andin vivoin psoriatic lesions. Increased TIG3 expression is correlated with decreased proliferation. TIG3 is expressed in a number of tissues, and expression is reduced in cancer cell lines and some primary tumors. In breast cancer cell lines, retinoid-dependent TIG3 induction is observed in lines that are growth suppressed by retinoids but not in nonresponsive lines. Transient over-expression of TIG3 in T47D or Chinese hamster ovary cells inhibits colony expansion. Finally, studies in 293 cells expressing TIG3 linked to an inducible promoter demonstrated decreased proliferation with increased TIG3 levels. These studies suggest that TIG3 may be a growth regulator that mediates some of the growth suppressive effects of retinoids.
Identification of a neuregulin and protein-tyrosine phosphatase response element in the nicotinic acetylcholine receptor ɛ subunit gene: Regulatory role of an Ets transcription factor
Authors: Sapru Mohan K. , Florance Sharon K. , Kirk Cassandra , Goldman Daniel
Published: July 2002

At the neuromuscular synapse, innervation induces endplate-specific expression of adult-type nicotinic acetylcholine receptors by selective expression of their subunit-encoding genes (α
2
βɛδ) in endplate-associated myonuclei. These genes are specifically regulated by protein-tyrosine phosphatase (PTPase) activity. In addition, neuregulin/acetylcholine-receptor-inducing activity, a nerve-derived factor that stimulates nicotinic acetylcholine receptor synthesis, induces adult-type specific ɛ subunit gene expression via activation of a Ras/mitogen-activated protein kinase pathway. However, the DNA regulatory elements and the binding proteins that mediate PTPase and neuregulin-dependent gene expression remain unknown. Herein we report that PTPase, neuregulin, and Ras-dependent regulation of the ɛ subunit gene map to a 15-bp promoter sequence. Interestingly, this same 15-bp sequence appears to be necessary for low ɛ subunit gene expression in extrajunctional regions of the muscle fiber. Site-directed mutagenesis of a putative Ets binding site located within this 15-bp sequence, reduced PTPase, neuregulin, and Ras-dependent regulation. Overexpression of the rat muscle Ets-2 transcription factor resulted in a sequence-specific induction of ɛ subunit promoter activity. Further, a dominant negative mutant of Ets-2 abolished neuregulin-dependent induction of ɛ subunit gene expression. Thus, these results indicate a crucial role for the 15-bp element in determining synapse-specific and neuregulin-mediated motor neuron control of ɛ subunit gene expression and suggest the participation of Ets transcription factor(s) in this control.
CD81 on B cells promotes interleukin 4 secretion and antibody production during T helper type 2 immune responses
Authors: Maecker Holden T. , Do Myoung-Sool , Levy Shoshana
Published: July 2002

Mice lacking CD81 (TAPA-1), a widely expressed tetraspanin molecule, have impaired antibody responses to protein antigens. This defect is specific to antigens that preferentially stimulate a T helper 2 response (ovalbumin or keyhole limpet hemocyanin in alum) and is only seen with T cell-dependent antigens. Absence of CD81 on B cells is sufficient to cause the defect. Also, antigen-specific interleukin (IL) 4 production is greatly reduced in the spleen and lymph nodes of CD81-null mice compared with heterozygous littermates. Thus, expression of CD81 on B cells is critical for inducing optimal IL-4 and antibody production during T helper 2 responses. These findings suggest that CD81 may interact with a ligand on T cells to signal IL-4 production. By using a soluble form of CD81 as a probe, a putative ligand for CD81 was identified on a subset of B and T cells. Two possible models for the interaction of CD81 on B cells with a potential ligand on either B or T cells are proposed.
T cell vaccination induces T cell receptor Vβ-specific Qa-1-restricted regulatory CD8+T cells
Authors: Jiang Hong , Kashleva Helena , Xu Li-Xing , Forman James , Flaherty Lorraine , Pernis Benvenuto , Braunstein Ned S. , Chess Leonard
Published: July 2002

Vaccination of mice with activated autoantigen-reactive CD4+T cells (T cell vaccination, TCV) has been shown to induce protection from the subsequent induction of a variety of experimental autoimmune diseases, including experimental allergic encephalomyelitis (EAE). Although the mechanisms involved in TCV-mediated protection are not completely known, there is some evidence that TCV induces CD8+regulatory T cells that are specific for pathogenic CD4+T cells. Previously, we demonstrated that, after superantigen administrationin vivo, CD8+T cells emerge that preferentially lyse and regulate activated autologous CD4+T cells in a T cell receptor (TCR) Vβ-specific manner. This TCR Vβ-specific regulation is not observed in β2-microglobulin-deficient mice and is inhibited,in vitro, by antibody to Qa-1. We now show that similar Vβ8-specific Qa-1-restricted CD8+T cells are also induced by TCV with activated CD4+Vβ8+T cells. These CD8+T cells specifically lyse murine or human transfectants coexpressing Qa-1 and murine TCR Vβ8. Further, CD8+T cell hybridoma clones generated from B10.PL mice vaccinated with a myelin basic protein-specific CD4+Vβ8+T cell clone specifically recognize other CD4+T cells and T cell tumors that express Vβ8 and the syngeneic Qa-1abut not the allogeneic Qa-1bmolecule. Thus, Vβ-specific Qa-1-restricted CD8+T cells are induced by activated CD4+T cells. We suggest that these CD8+T cells may function to specifically regulate activated CD4+T cells during immune responses.
CMS: An adapter molecule involved in cytoskeletal rearrangements
Authors: Kirsch Kathrin H. , Georgescu Maria-Magdalena , Ishimaru Satoshi , Hanafusa Hidesaburo
Published: July 2002

Cas ligand with multiple Src homology (SH) 3 domains (CMS) is an ubiquitously expressed signal transduction molecule that interacts with the focal adhesion protein p130
Cas
. CMS contains three SH3 in its NH
2
terminus and proline-rich sequences in its center region. The latter sequences mediate the binding to the SH3 domains of p130
Cas
, Src-family kinases, p85 subunit of phosphatidylinositol 3-kinase, and Grb2. The COOH-terminal region contains putative actin binding sites and a coiled-coil domain that mediates homodimerization of CMS. CMS is a cytoplasmic protein that colocalizes with F-actin and p130
Cas
to membrane ruffles and leading edges of cells. Ectopic expression of CMS in COS-7 cells resulted in alteration in arrangement of the actin cytoskeleton. We observed a diffuse distribution of actin in small dots and less actin fiber formation. Altogether, these features suggest that CMS functions as a scaffolding molecule with a specialized role in regulation of the actin cytoskeleton.
MSF
(MLL septin-like fusion), a fusion partner gene of
MLL,
in a therapy-related acute myeloid leukemia with a t(11;17)(q23;q25)
Authors: Osaka Mitsuhiko , Rowley Janet D. , Zeleznik-Le Nancy J.
Published: July 2002

MLL
(
ALL1, Htrx, HRX
), which is located on chromosome band 11q23, frequently is rearranged in patients with therapy-related acute myeloid leukemia who previously were treated with DNA topoisomerase II inhibitors. In this study, we have identified a fusion partner of
MLL
in a 10-year-old female who developed therapy-related acute myeloid leukemia 17 months after treatment for Hodgkin’s disease. Leukemia cells of this patient had a t(11;17)(q23;q25), which involved
MLL
as demonstrated by Southern blot analysis. The partner gene was cloned from cDNA of the leukemia cells by use of a combination of adapter reverse transcriptase–PCR, rapid amplification of 5′ cDNA ends, and
blast
database analysis to identify expressed sequence tags. The full-length cDNA of 2.8 kb was found to be an additional member of the septin family, therefore it was named
MSF
(MLL septin-like fusion). Members of the septin family conserve the GTP binding domain, localize in the cytoplasm, and interact with cytoskeletal filaments. A major 4-kb transcript of
MSF
was expressed ubiquitously; a 1.7-kb transcript was found in most tissues. An additional 3-kb transcript was found only in hematopoietic tissues. By amplification with
MLL
exon 5 forward primer and reverse primers in
MSF
, the appropriately sized products were obtained.
MSF
is highly homologous to
hCDCrel-1
, which is a partner gene of
MLL
in leukemias with a t(11;22)(q23;q11.2). Further analysis of
MSF
may help to delineate the function of
MLL
partner genes in leukemia, particularly in therapy-related leukemia.
Implications of immune-to-brain communication for sickness and pain
Authors: Watkins Linda R. , Maier Steven F.
Published: July 2002

This review presents a view of hyperalgesia and allodynia not
typical of the field as a whole. That is, exaggerated pain is presented
as one of many natural consequences of peripheral infection and injury.
The constellation of changes that results from such immune challenges
is called the sickness response. This sickness response results from
immune-to-brain communication initiated by proinflammatory cytokines
released by activated immune cells. In response to signals it receives
from the immune system, the brain orchestrates the broad array of
physiological, behavioral, and hormonal changes that comprise the
sickness response. The neurocircuitry and neurochemistry of
sickness-induced hyperalgesia are described. One focus of this
discussion is on the evidence that spinal cord microglia and astrocytes
are key mediators of sickness-induced hyperalgesia. Last, evidence is
presented that hyperalgesia and allodynia also result from direct
immune activation, rather than neural activation, of these same spinal
cord glia. Such glial activation is induced by viruses such as HIV-1
that are known to invade the central nervous system. Implications of
exaggerated pain states created by peripheral and central immune
activation are discussed.
Mucolipidosis IV consists of one complementation group
Authors: Goldin Ehud , Cooney Adele , Kaneski Christine R. , Brady Roscoe O. , Schiffmann Raphael
Published: July 2002

Mucolipidosis IV (MLIV) is an autosomal recessive disorder of unknown etiology characterized by severe visual impairment and psychomotor retardation. Recently, there has been considerable interest in positional cloning of the MLIV gene. It is unknown whether MLIV is a genetically homogenous disorder. In this paper, we present experiments that determined whether the MLIV phenotype in fibroblasts could be corrected by fusing normal cells to MLIV cells and fusing fibroblasts from pairs of patients. All of our MLIV patients fulfilled several diagnostic criteria that we developed. In addition, we found high sensitivity to chloroquine in cultured fibroblasts from MLIV patients. We found that normal cells corrected the MLIV phenotype. Fusion products of normal and MLIV fibroblasts, but not MLIV fibroblasts themselves, were relatively protected against chloroquine selection. In addition, 74% of the normal-to-patient fusion products had reduced levels or total loss of MLIV characteristic autofluorescence. However, there was no complementation of the phenotype in fibroblast cultures from any of our MLIV patients, including those of non-Jewish ancestry. In fusion products of MLIV cultures from 24 patients, 90–100% of the cells remained autofluorescent. These results indicate that all of our known MLIV patients, regardless of ancestry or severity of the developmental defect, have a single mutated gene.
Inhibition of NF-κB potentiates amyloid β-mediated neuronal apoptosis
Authors: Kaltschmidt Barbara , Uherek Martin , Wellmann Henning , Volk Benedikt , Kaltschmidt Christian
Published: July 2002

One mechanism leading to neurodegeneration during Alzheimer’s disease (AD) is amyloid β peptide (Aβ) neurotoxicity. Aβ elicits in cultured central nervous system neurons a biphasic response: a low-dose neurotrophic response and a high-dose neurotoxic response. Previously we reported that NF-κB is activated by low doses of Aβ only. Here we show that NF-κB activation leads to neuroprotection. In primary neurons we found that a pretreatment with 0.1 μM Aβ-(1–40) protects against neuronal death induced with 10 μM Aβ-(1–40). As a known neuroprotective agent we next analyzed the effect of tumor necrosis factor α (TNF-α). Maximal activation of NF-κB was found with 2 ng/ml TNF-α. Pretreatment with TNF-α protected cerebellar granule cells from cell death induced by 10 μM Aβ-(1–40). This protection is described by an inverted U-shaped dose response and is maximal with a NF-κB-activating dose. The molecular specificity of this protective effect was analyzed by specific blockade of NF-κB activation. Overexpression of a transdominant negative IκB-α blocks NF-κB activation and potentiates Aβ-mediated neuronal apoptosis. Our findings show that activation of NF-κB is the underlying mechanism of the neuroprotective effect of low-dose Aβ and TNF-α. In accordance with these
in vitro
data we find that nuclear NF-κB immunoreactivity around various plaque stages of AD patients is reduced in comparison to age-matched controls. Taken together these data suggest that pharmacological NF-κB activation may be a useful approach in the treatment of AD and related neurodegenerative disorders.
Intra-tRNA distance measurements for nucleocapsid proteindependent tRNA unwinding during priming of HIV reverse transcription
Authors: Chan Barden , Weidemaier Kristin , Yip Wai-Tak , Barbara Paul F. , Musier-Forsyth Karin
Published: July 2002

We report here the direct measurement of intra-tRNA distances during annealing of the tRNA primer to the HIV RNA genome. This key step in the initiation of retroviral reverse transcription involves hybridization of one strand of the acceptor arm of a specific lysine tRNA to the primer binding site on the RNA genome. Although the mechanism of tRNA unwinding and annealing is not known, previous studies have shown that HIV nucleocapsid protein (NC) greatly accelerates primer/template binary complex formation
in vitro
. An open question is whether NC alone unwinds the primer or whether unwinding by NC requires the RNA genome. We monitored the annealing process in solution by using fluorescence resonance energy transfer (FRET). Distance measurements demonstrate unequivocally that the tRNA acceptor stem is not substantially unwound by NC in the absence of the RNA genome, that is, unwinding is not separable from hybridization. Moreover, FRET measurements show that both heat- and NC-mediated annealing result in an ≈40-Å increase in the separation of the two ends of the tRNA acceptor arm on binding to the template. This large increase in separation of the two ends suggests a complete displacement of the nonhybridized strand of the acceptor stem in the initiation complex.
In situ
detection of the hypermethylation-induced inactivation of the
p16
gene as an early event in oncogenesis
Authors: Nuovo Gerard J. , Plaia Todd W. , Belinsky Steven A. , Baylin Stephen B. , Herman James G.
Published: July 2002

We have developed a technique, methylation-specific PCR
in situ
hybridization (MSP-ISH), which allows for the methylation status of specific DNA sequences to be visualized in individual cells. We use MSP-ISH to monitor the timing and consequences of aberrant hypermethylation of the
p16
tumor suppresser gene during the progression of cancers of the lung and cervix. Hypermethylation of
p16
was localized only to the neoplastic cells in both
in situ
lesions and invasive cancers, and was associated with loss of p16 protein expression. MSP-ISH allowed us to dissect the surprising finding that
p16
hypermethylation occurs in cervical carcinoma. This tumor is associated with infection of the oncogenic human papillomavirus, which expresses a protein, E7, that inactivates the retinoblastoma (Rb) protein. Thus, simultaneous Rb and
p16
inactivation would not be needed to abrogate the critical cyclin D–Rb pathway. MSP-ISH reveals that
p16
hypermethylation occurs heterogeneously within early cervical tumor cell populations that are separate from those expressing viral E7 transcripts. In advanced cervical cancers, the majority of cells have a hypermethylated
p16
, lack p16 protein, but no longer express E7. These data suggest that p16 inactivation is selected as the most effective mechanism of blocking the cyclin D–Rb pathway during the evolution of an invasive cancer from precursor lesions. These studies demonstrate that MSP-ISH is a powerful approach for studying the dynamics of aberrant methylation of critical tumor suppressor genes during tumor evolution.
Structure of the HIV-1 integrase catalytic domain complexed with an inhibitor: A platform for antiviral drug design
Authors: Goldgur Yehuda , Craigie Robert , Cohen Gerson H. , Fujiwara Tamio , Yoshinaga Tomokazu , Fujishita Toshio , Sugimoto Hirohiko , Endo Takeshi , Murai Hitoshi , Davies David R.
Published: July 2002

HIV integrase, the enzyme that inserts the viral DNA into the host chromosome, has no mammalian counterpart, making it an attractive target for antiviral drug design. As one of the three enzymes produced by HIV, it can be expected that inhibitors of this enzyme will complement the therapeutic use of HIV protease and reverse transcriptase inhibitors. We have determined the structure of a complex of the HIV-1 integrase core domain with a novel inhibitor, 5ClTEP, 1-(5-chloroindol-3-yl)-3-hydroxy-3-(2
H
-tetrazol-5-yl)-propenone, to 2.1-Å resolution. The inhibitor binds centrally in the active site of the integrase and makes a number of close contacts with the protein. Only minor changes in the protein accompany inhibitor binding. This inhibitor complex will provide a platform for structure-based design of an additional class of inhibitors for antiviral therapy.
Torpor in mice is induced by both leptin-dependent and -independent mechanisms
Authors: Gavrilova Oksana , Leon Lisa R. , Marcus-Samuels Bernice , Mason Mark M. , Castle Arthur L. , Refetoff Samuel , Vinson Charles , Reitman Marc L.
Published: July 2002

We tested the effect of chronic leptin treatment on fasting-induced torpor in leptin-deficient A-ZIP/F-1 and
ob/ob
mice. A-ZIP/F-1 mice have virtually no white adipose tissue and low leptin levels, whereas
ob/ob
mice have an abundance of fat but no leptin. These two models allowed us to examine the roles of adipose tissue and leptin in the regulation of entry into torpor. Torpor is a short-term hibernation-like state that allows conservation of metabolic fuels. We first characterized the A-ZIP/F-1 animals, which have a 10-fold reduction in total body triglyceride stores. Upon fasting, A-ZIP/F-1 mice develop a lower metabolic rate and decreased plasma glucose, insulin, and triglyceride levels, with no increase in free fatty acids or β-hydroxybutyrate. Unlike control mice, by 24 hr of fasting, they have nearly exhausted their triglycerides and are catabolizing protein. To conserve energy supplies during fasting, A-ZIP/F-1 (but not control) mice entered deep torpor, with a minimum core body temperature of 24°C, 2°C above ambient. In
ob/ob
mice, fasting-induced torpor was completely reversed by leptin treatment. In contrast, neither leptin nor thyroid hormone prevented torpor in A-ZIP/F-1 mice. These data suggest that there are at least two signals for entry into torpor in mice, a low leptin level and another signal that is independent of leptin and thyroid hormone levels. Studying rodent torpor provides insight into human torpor-like states such as near drowning in cold water and induced hypothermia for surgery.
In vivo
analysis of the 3′ untranslated region of the hepatitis C virus after
in vitro
mutagenesis of an infectious cDNA clone
Authors: Yanagi Masayuki , St. Claire Marisa , Emerson Suzanne U. , Purcell Robert H. , Bukh Jens
Published: July 2002

Large sections of the 3′ untranslated region (UTR) of hepatitis C virus (HCV) were deleted from an infectious cDNA clone, and the RNA transcripts from seven deletion mutants were tested sequentially for infectivity in a chimpanzee. Mutants lacking all or part of the 3′ terminal conserved region or the poly(U–UC) region were unable to infect the chimpanzee, indicating that both regions are critical for infectivity
in vivo
. However, the third region, the variable region, was able to tolerate a deletion that destroyed the two putative stem–loop structures within this region. Mutant VR-24 containing a deletion of the proximal 24 nt of the variable region of the 3′ UTR was viable in the chimpanzee and seemed to replicate as well as the undeleted parent virus. The chimpanzee became viremic 1 week after inoculation with mutant VR-24, and the HCV genome titer increased over time during the early acute infection. Therefore, the poly(U–UC) region and the conserved region, but not the variable region, of the 3′ UTR seem to be critical for
in vivo
infectivity of HCV.
Functional repair of motor endplates after botulinum neurotoxin type A poisoning: Biphasic switch of synaptic activity between nerve sprouts and their parent terminals
Authors: de Paiva Anton , Meunier Frédéric A. , Molgó Jordi , Aoki K. Roger , Dolly J. Oliver
Published: July 2002

Blockade of acetylcholine release by botulinum neurotoxin type A at the neuromuscular junction induces the formation of an extensive network of nerve-terminal sprouts. By repeated
in vivo
imaging of
N
-(3-triethyl ammonium propyl)-4-(4-(dibutylamino)styryl) pyridinium dibromide uptake into identified nerve endings of the mouse sternomastoid muscle after a single intramuscular injection of the toxin, inhibition of stimulated uptake of the dye at the terminals was detected within a few days, together with an increase in staining of the newly formed sprouts. After 28 days, when nerve stimulation again elicited muscle contraction, regulated vesicle recycling occurred only in the sprouts [shown to contain certain soluble
N
-ethylmaleimide-sensitive factor attachment proteins (SNAREs) and to abut acetylcholine receptors] and not at the parent terminals. Therefore, only these sprouts could be responsible for nerve–muscle transmission at this time. However, a second, distinct phase of the rehabilitation process followed with a return of vesicle turnover to the original terminals, accompanied by an elimination of the by then superfluous sprouts. This extension and later removal of “functional” sprouts indicate their fundamental importance in the repair of paralyzed endplates, a finding with ramifications for the vital process of nerve regeneration.
Double-stranded RNA specifically disrupts gene expression during planarian regeneration
Authors: Sánchez Alvarado Alejandro , Newmark Phillip A.
Published: July 2002

Metazoan regeneration is one of the least understood fundamental problems of biology. The lack of progress in understanding this phenomenon at the molecular level has been due to the poor regenerative abilities of the genetic organisms used for developmental studies, as well as the difficulties encountered with molecular and genetic manipulations of the commonly studied vertebrate models (the urodele amphibians). Here, we demonstrate that introduction of double-stranded RNA selectively abrogates gene function in planarians, a classic model of regeneration. The ability to eliminate gene function in a regenerating organism such as the planarian overcomes previous experimental limitations and opens the study of animal regeneration to unprecedented levels of molecular detail.
Accumulation of protease-resistant prion protein (PrP) and apoptosis of cerebellar granule cells in transgenic mice expressing a PrP insertional mutation
Authors: Chiesa Roberto , Drisaldi Bettina , Quaglio Elena , Migheli Antonio , Piccardo Pedro , Ghetti Bernardino , Harris David A.
Published: July 2002

We have generated lines of transgenic mice that express a mutant prion protein (PrP) containing 14 octapeptide repeats whose human homologue is associated with an inherited prion dementia. These mice develop a neurological illness with prominent ataxia at 65 or 240 days of age, depending on whether the transgene array is, respectively, homozygous or hemizygous. Starting from birth, mutant PrP is converted into a protease-resistant and detergent-insoluble form that resembles the scrapie isoform of PrP, and this form accumulates dramatically in many brain regions throughout the lifetime of the mice. As PrP accumulates, there is massive apoptosis of granule cells in the cerebellum. Our analysis provides important insights into the molecular pathogenesis of inherited prion disorders in humans.
Testosterone reduces neuronal secretion of Alzheimer's β-amyloid peptides
Authors: Gouras Gunnar K. , Xu Huaxi , Gross Rachel S. , Greenfield Jeffrey P. , Hai Bing , Wang Rong , Greengard Paul
Published: July 2002

Alzheimer's disease (AD) is characterized by the age-related deposition of β-amyloid (Aβ) 40/42 peptide aggregates in vulnerable brain regions. Multiple levels of evidence implicate a central role for Aβ in the pathophysiology of AD. Aβ peptides are generated by the regulated cleavage of an ≈700-aa Aβ precursor protein (βAPP). Full-length βAPP can undergo proteolytic cleavage either within the Aβ domain to generate secreted sβAPPα or at the N- and C-terminal domain(s) of Aβ to generate amyloidogenic Aβ peptides. Several epidemiological studies have reported that estrogen replacement therapy protects against the development of AD in postmenopausal women. We previously reported that treating cultured neurons with 17β-estradiol reduced the secretion of Aβ40/42 peptides, suggesting that estrogen replacement therapy may protect women against the development of AD by regulating βAPP metabolism. Increasing evidence indicates that testosterone, especially bioavailable testosterone, decreases with age in older men and in postmenopausal women. We report here that treatment with testosterone increases the secretion of the nonamyloidogenic APP fragment, sβAPPα, and decreases the secretion of Aβ peptides from N2a cells and rat primary cerebrocortical neurons. These results raise the possibility that testosterone supplementation in elderly men may be protective in the treatment of AD.