bluuebunny/crossref_metadata_2025 huggingface dataset:
Early-life exposure to endotoxin alters hypothalamic–pituitary–adrenal function and predisposition to inflammation
Authors: Shanks Nola , Windle Richard J. , Perks Paula A. , Harbuz Michael S. , Jessop David S. , Ingram Colin D. , Lightman Stafford L.
Published: July 2002

We have investigated whether exposure to Gram-negative bacterial endotoxin in early neonatal life can alter neuroendocrine and immune regulation in adult animals. Exposure of neonatal rats to a low dose of endotoxin resulted in long-term changes in hypothalamic–pituitary–adrenal (HPA) axis activity, with elevated mean plasma corticosterone concentrations that resulted from increased corticosterone pulse frequency and pulse amplitude. In addition to this marked effect on the development of the HPA axis, neonatal endotoxin exposure had long-lasting effects on immune regulation, including increased sensitivity of lymphocytes to stress-induced suppression of proliferation and a remarkable protection from adjuvant-induced arthritis. These findings demonstrate a potent and long-term effect of neonatal exposure to inflammatory stimuli that can program major changes in the development of both neuroendocrine and immunological regulatory mechanisms.
Association and regulation of casein kinase 2 activity by adenomatous polyposis coli protein
Authors: Homma Miwako Kato , Li Dongxia , Krebs Edwin G. , Yuasa Yasuhito , Homma Yoshimi
Published: July 2002

Mutations in the adenomatous polyposis coli (
APC
) gene are responsible for familial adenomatous polyposis coli and also sporadic colorectal cancer development. By using antibodies raised against the N-terminal region of APC protein, we have detected the variable masses of endogenous APC proteins in individual cell lines established from human colorectal carcinomas caused by nonsense mutations of the gene. Phosphorylation of immunoprecipitates of full-length and truncated APC were observed in
in vitro
kinase reaction, indicating association of APC with protein kinase activity. The kinase activity complexed with APC was sensitive to heparin and used GTP as phosphoryl donor, suggesting an involvement of casein kinase 2 (CK2). Both CK2α- and β-subunits were found to associate with APC in immunoprecipitates as well as in pull-down assays, with preferential interaction of APC with tetrameric CK2 holoenzyme. In synchronized cell populations, the association of APC with CK2 was cell cycle dependent, with the highest association in G
2
/M. Unexpectedly, APC immunoprecipitates containing full-length APC protein inhibited CK2
in vitro
, whereas immunoprecipitates of truncated APC had little effect. This was confirmed by using recombinant APC, and the inhibitory region was localized to the C terminus of APC between residues 2086 and 2394. Overexpression of this fragment in SW480 cells suppressed cell proliferation rates as well as tumorigenesis. These results demonstrate a previously uncharacterized functional interaction between the tumor suppressor protein APC and CK2 and suggest that growth-inhibitory effects of APC may be regulated by inhibition of CK2.
ATP6S1 elicits potent humoral responses associated with immune-mediated tumor destruction
Authors: Hodi F. Stephen , Schmollinger Jan C. , Soiffer Robert J. , Salgia Ravi , Lynch Thomas , Ritz Jerome , Alyea Edwin P. , Yang Jihong , Neuberg Donna , Mihm Martin , Dranoff Glenn
Published: July 2002

An important goal of cancer immunology is the identification of antigens associated with tumor destruction. Vaccination with irradiated tumor cells engineered to secrete granulocyte/macrophage colony-stimulating factor (GM-CSF) generates potent, specific, and long-lasting antitumor immunity in multiple murine tumor models. A phase I clinical trial of this vaccination strategy in patients with advanced melanoma demonstrated the consistent induction of dense CD4
+
and CD8
+
T lymphocyte and plasma cell infiltrates in distant metastases, resulting in extensive tumor destruction, fibrosis, and edema. Antimelanoma antibody and cytotoxic T cell responses were associated with tumor cell death. To characterize the targets of these responses, we screened an autologous cDNA expression library prepared from a densely infiltrated metastasis with postvaccination sera from a long-term responding patient. High-titer IgG antibodies detected ATP6S1, a putative accessory unit of the vacuolar H
+
–ATPase complex. A longitudinal analysis of this patient revealed an association between the vaccine-induced increase in antibodies to ATP6S1 and tumor destruction. Three additional vaccinated melanoma patients and three metastatic non-small cell lung carcinoma patients vaccinated with autologous GM-CSF-secreting tumor cells similarly showed a correlation between humoral responses to ATP6S1 and tumor destruction. Moreover, a chronic myelogenous leukemia patient who experienced a complete remission after CD4
+
donor lymphocyte infusions also developed high-titer antibodies to ATP6S1. Lastly, vaccination with GM-CSF-secreting B16 melanoma cells stimulated high-titer antibodies to ATPS1 in a murine model. Taken together, these findings demonstrate that potent humoral responses to ATP6S1 are associated with immune-mediated destruction of diverse tumors.
Variant-type PML-RARα fusion transcript in acute promyelocytic leukemia: Use of a cryptic coding sequence from intron 2 of the
RAR
α gene and identification of a new clinical subtype resistant to retinoic acid therapy
Authors: Gu Bai-Wei , Xiong Hui , Zhou Yan , Chen Bing , Wang Li , Dong Shuo , Yu Zhi-Yuan , Lu Ling-Feng , Zhong Ming , Yin Hai-Feng , Zhu Gen-Feng , Huang Wei , Ren Shuang-Xi , Gallagher Robert E. , Waxman Samuel , Chen Guo-Qiang , Wang Zhu-Gang , Chen Zhu , Fu Gang , Chen Sai-Juan
Published: July 2002

The physiologic actions of retinoic acids (RAs) are mediated through RA receptors (RARs) and retinoid X receptors (RXRs). The
RAR
α gene has drawn particular attention because it is the common target in all chromosomal translocations in acute promyelocytic leukemia (APL), a unique model in cancer research that responds to the effect of RA. In the great majority of patients with APL,
RAR
α is fused to the
PML
gene as a result of the t(15;17) translocation. Three distinct types of
PML-RAR
α transcripts, long (L), short (S), and variant (V), were identified. The V-type is characterized by truncation of exon 6 of
PML
and in some cases by the insertion of a variable “spacer” sequence between the truncated
PML
and
RAR
α mRNA fusion partners, although the precise mechanisms underlying formation of the V-type transcript remain unclear. To get further insights into the molecular basis of the t(15;17), we sequenced the entire genomic DNA region of
RAR
α. Of note, all previously reported “spacer” sequences in V-type transcripts were found in intron 2 of the
RAR
α gene and most of these sequences were flanked by gt splice donor sites. In most cases, these “cryptic” coding sequences maintained the ORF of the chimeric transcript. Interestingly, two cases with a relatively long spacer sequence showed APL cellular and clinical resistance to RA treatment. In these cases, the aberrant V-type
PML-RAR
α protein displayed increased affinity to the nuclear corepressor protein SMRT, providing further evidence that RA exerts the therapeutic effect on APL through modulation of the RAR–corepressor interaction. Finally, among patients with the L- or S-type PML-RARα fusion transcript, some consensus motifs were identified at the hotspots of the chromosome 17q breakpoints within intron 2 of
RAR
α, strengthening the importance of this intron in the molecular pathogenesis of APL.
Submucosal gland secretions in airways from cystic fibrosis patients have normal [Na+] and pH but elevated viscosity
Authors: Jayaraman Sujatha , Joo Nam Soo , Reitz Bruce , Wine Jeffrey J. , Verkman A. S.
Published: July 2002

Fluid and macromolecule secretion by submucosal glands in mammalian airways is believed to be important in normal airway physiology and in the pathophysiology of cystic fibrosis (CF). Anin situfluorescence method was applied to measure the ionic composition and viscosity of freshly secreted fluid from airway glands. Fragments of human large airways obtained at the time of lung transplantation were mounted in a humidified perfusion chamber and the mucosal surface was covered by a thin layer of oil. Individual droplets of secreted fluid were microinjected with fluorescent indicators for measurement of [Na+], [Cl−], and pH by ratio imaging fluorescence microscopy and viscosity by fluorescence recovery after photobleaching. After carbachol stimulation, 0.1–0.5 μl of fluid accumulated in spherical droplets at gland orifices in ≈3–5 min. In gland fluid from normal human airways, [Na+] was 94 ± 8 mM, [Cl−] was 92 ± 12 mM, and pH was 6.97 ± 0.06 (SE,n= 7 humans, more than five glands studied per sample). Apparent fluid viscosity was 2.7 ± 0.3-fold greater than that of saline. Neither [Na+] nor pH differed in gland fluid from CF airways, but viscosity was significantly elevated by ≈2-fold compared to normal airways. These results represent the first direct measurements of ionic composition and viscosity in uncontaminated human gland secretions and indicate similar [Na+], [Cl−], and pH to that in the airway surface liquid. The elevated gland fluid viscosity in CF may be an important factor promoting bacterial colonization and airway disease.
Altered lymphocyte responses and cytokine production in mice deficient in the X-linked lymphoproliferative disease geneSH2D1A/DSHP/SAP
Authors: Czar Michael J. , Kersh Ellen N. , Mijares Lilia A. , Lanier Gibson , Lewis Jennifer , Yap George , Chen Amy , Sher Alan , Duckett Colin S. , Ahmed Rafi , Schwartzberg Pamela L.
Published: July 2002

We have introduced a targeted mutation inSH2D1A/DSHP/SAP, the gene responsible for the human genetic disorder X-linked lymphoproliferative disease (XLP). SLAM-associated protein (SAP)-deficient mice had normal lymphocyte development, but on challenge with infectious agents, recapitulated features of XLP. Infection of SAP−mice with lymphocyte choriomeningitis virus (LCMV) orToxoplasma gondiiwas associated with increased T cell activation and IFN-γ production, as well as a reduction of Ig-secreting cells. Anti-CD3-stimulated splenocytes from uninfected SAP−mice produced increased IFN-γ and decreased IL-4, findings supported by decreased serum IgE levelsin vivo. The Th1 skewing of these animals suggests that cytokine misregulation may contribute to phenotypes associated with mutation ofSH2D1A/SAP.
Sensitive detection of DNA polymorphisms by the serial invasive signal amplification reaction
Authors: Hall Jeff G. , Eis Peggy S. , Law Scott M. , Reynaldo Luis P. , Prudent James R. , Marshall David J. , Allawi Hatim T. , Mast Andrea L. , Dahlberg James E. , Kwiatkowski Robert W. , de Arruda Monika , Neri Bruce P. , Lyamichev Victor I.
Published: July 2002

The invasive signal amplification reaction has been previously developed for quantitative detection of nucleic acids and discrimination of single-nucleotide polymorphisms. Here we describe a method that couples two invasive reactions into a serial isothermal homogeneous assay using fluorescence resonance energy transfer detection. The serial version of the assay generates more than 10
7
reporter molecules for each molecule of target DNA in a 4-h reaction; this sensitivity, coupled with the exquisite specificity of the reaction, is sufficient for direct detection of less than 1,000 target molecules with no prior target amplification. Here we present a kinetic analysis of the parameters affecting signal and background generation in the serial invasive signal amplification reaction and describe a simple kinetic model of the assay. We demonstrate the ability of the assay to detect as few as 600 copies of the methylene tetrahydrofolate reductase gene in samples of human genomic DNA. We also demonstrate the ability of the assay to discriminate single base differences in this gene by using 20 ng of human genomic DNA.
The architecture of the human Rad54–DNA complex provides evidence for protein translocation along DNA
Authors: Ristic Dejan , Wyman Claire , Paulusma Coen , Kanaar Roland
Published: July 2002

Proper maintenance and duplication of the genome require accurate recombination between homologous DNA molecules. In eukaryotic cells, the Rad51 protein mediates pairing between homologous DNA molecules. This reaction is assisted by the Rad54 protein. To gain insight into how Rad54 functions, we studied the interaction of the human Rad54 (hRad54) protein with double-stranded DNA. We have recently shown that binding of hRad54 to DNA induces a change in DNA topology. To determine whether this change was caused by a protein-constrained change in twist, a protein-constrained change in writhe, or the introduction of unconstrained plectonemic supercoils, we investigated the hRad54–DNA complex by scanning force microscopy. The architecture of the observed complexes suggests that movement of the hRad54 protein complex along the DNA helix generates unconstrained plectonemic supercoils. We discuss how hRad54-induced superhelical stress in the target DNA may function to facilitate homologous DNA pairing by the hRad51 protein directly. In addition, the induction of supercoiling by hRad54 could stimulate recombination indirectly by displacing histones and/or other proteins packaging the DNA into chromatin. This function of DNA translocating motors might be of general importance in chromatin metabolism.
A dual role for the kinase-like domain of the tyrosine kinase Tyk2 in interferon-α signaling
Authors: Yeh Tammie C. , Dondi Elisabetta , Uzé Gilles , Pellegrini Sandra
Published: July 2002

Tyrosine kinases of the Janus kinase family initiate cellular responses through their association with receptors for α-helical cytokines. In addition to a tyrosine kinase domain, these enzymes possess a kinase-like (KL) domain, whose function remains elusive. To investigate the role of the KL domain of Tyk2 in interferon-α/β signaling, we transfected a library of Tyk2 cDNAs containing random point mutations in KL into Tyk2-negative cells and selected for loss-of-function Tyk2 mutants. Four such mutants, V584D, G596V, H669P, and R856G, were identified through this screen. Like the wild-type Tyk2, the mutant proteins were able to sustain the level of IFNAR1 receptor protein. However, all four mutants were incapable of restoring high-affinity interferon-α binding in Tyk2-negative cells and were also catalytically impaired, even when transiently overexpressed. Interferon-α induced phosphorylation, and gene expression could be detected in V584D- or G596V-expressing cells, but not in H669P- or R856G-expressing cells. Furthermore, H669P and R856G proteins were constitutively highly phosphorylated. All together, our findings demonstrate that an intact KL domain is essential for the intrinsic catalytic activity of Tyk2 and for the establishment of a high-affinity interferon-α receptor complex.
Comparative expressed sequence hybridization to chromosomes for tumor classification and identification of genomic regions of differential gene expression
Authors: Lu Yong-Jie , Williamson Daniel , Clark Jeremy , Wang Rubin , Tiffin Nicki , Skelton Lorraine , Gordon Tony , Williams Richard , Allan Barry , Jackman Ann , Cooper Colin , Pritchard-Jones Kathy , Shipley Janet
Published: July 2002

Altered expression of genes can have phenotypic consequences in cancer development and treatment, developmental abnormalities, and differentiation processes. Here we describe a rapid approach, termed comparative expressed sequence hybridization (CESH), which gives a genome-wide view of relative expression patterns within tissues according to chromosomal location. No prior knowledge of genes or cloning is required, and minimal amounts of tissue can be used. Expression profiles are achieved in a manner similar to the identification of chromosomal imbalances by comparative genomic hybridization analysis. The approach is demonstrated to indicate a chromosomal region that harbors overexpressed genes that may be associated with a drug-resistant phenotype. In addition, known and new regions of differential gene expression in both normal tissues and tumor samples from the soft tissue sarcoma group of rhabdomyosarcoma (RMS) are indicated. These regions included 2p24; overexpression of
MYCN
at 2p24 was confirmed by quantitative reverse transcription–PCR for all of the alveolar RMS cases and did not necessarily correspond to genomic amplification. Evidence including region specific microarray analysis indicated that overexpression of several genes from a region may be required for detection by CESH. This evidence is consistent with clusters of functionally related genes and mechanisms that affect the expression of a number of genes at a particular genomic location. The distinctive CESH profiles demonstrated in different subtypes of RMS show potential for tumor classification.
The infected cell protein 0 of herpes simplex virus 1 dynamically interacts with proteasomes, binds and activates the cdc34 E2 ubiquitin-conjugating enzyme, and possesses
in vitro
E3 ubiquitin ligase activity
Authors: Van Sant Charles , Hagglund Ryan , Lopez Pascal , Roizman Bernard
Published: July 2002

The infected cell protein 0 (ICP0) of herpes simplex virus 1, a promiscuous transactivator shown to enhance the expression of genes introduced into cells by infection or transfection, interacts with numerous cellular proteins and has been linked to the disruption of ND10 and degradation of several proteins. ICP0 contains a RING finger domain characteristic of a class of E3 ubiquitin ligases. We report that: (
i
) in infected cells, ICP0 interacts dynamically with proteasomes and is bound to proteasomes in the presence of the proteasome inhibitor MG132. Also in infected cells, cdc34, a polyubiquitinated E2 ubiquitin-conjugating enzyme, exhibits increased ICP0-dependent dynamic interaction with proteasomes. (
ii
) In an
in vitro
substrate-independent ubiquitination system, the RING finger domain encoded by exon 2 of ICP0 binds cdc34, whereas the carboxyl-terminal domain of ICP0 functions as an E3 ligase independent of the RING finger domain. The results indicate that ICP0 can act as a unimolecular E3 ubiquitin ligase and that it promotes ubiquitin-protein ligation and binds the E2 cdc34. It differs from other unimolecular E3 ligases in that the domain containing the RING finger binds E2, whereas the ligase activity maps to a different domain of the protein. The results also suggest that ICP0 shuttles between nucleus and cytoplasm as a function of its dynamic interactions with proteasomes.
Polo-like kinase is required for the fragmentation of pericentriolar Golgi stacks during mitosis
Authors: Sütterlin Christine , Lin Chin-Yo , Feng Yang , Ferris Douglas K. , Erikson Raymond L. , Malhotra Vivek
Published: July 2002

The pericentriolar stacks of Golgi cisternae undergo extensive reorganization during mitosis in mammalian cells. GM130 and GRASP65 (Golgi reassembly stacking protein of 65 kDa) are Golgi-associated proteins that are targets of mitotic kinases, and they have also been implicated in the reorganization of the Golgi structure during cell division. Previous studies have reported that mitogen-activated protein kinase kinase-1 (MEK1) and Cdc2 protein kinases are involved in these dynamic changes in the Golgi structure. More recently, the mitotic polo-like kinase (Plk) has been shown to interact with and phosphorylate GRASP65. Here, we provide evidence that Plk is involved in the mitosis-specific fragmentation of the Golgi apparatus. The addition of kinase-defective Plk or immunodepletion of Plk disrupts the fragmentation process. Furthermore, Golgi fragmentation is inhibited by the addition of either full-length or truncated GRASP65. These findings suggest that phosphorylation of GRASP65 by Plk may be a critical event in the reorganization of the Golgi structure during mitosis.
Function of
bicoid
and
hunchback
homologs in the basal cyclorrhaphan fly
Megaselia
(Phoridae)
Authors: Stauber Michael , Taubert Heike , Schmidt-Ott Urs
Published: July 2002

The
Drosophila
gene
bicoid
functions
at the beginning of a gene cascade that specifies anterior structures
in the embryo. Its transcripts are localized at the anterior pole of
the oocyte, giving rise to a Bicoid protein gradient, which regulates
the spatially restricted expression of target genes along the
anterior–posterior axis of the embryo in a concentration-dependent
manner. The morphogen function of Bicoid requires the coactivity of the
zinc finger transcription factor Hunchback, which is expressed in a
Bicoid-dependent fashion in the anterior half of the embryo. Whereas
hunchback
is conserved throughout insects,
bicoid
homologs are known only from cyclorrhaphan flies.
Thus far, identification of
hunchback
and
bicoid
homologs rests only on sequence comparison. In
this study, we used double-stranded RNA interference (RNAi) to address
the function of
bicoid
and
hunchback
homologs in embryos of the lower cyclorrhaphan fly
Megaselia
abdita
(Phoridae).
Megaselia-hunchback
RNAi
causes
hunchback
-like phenotypes as observed in
Drosophila
, but
Megaselia-bicoid
RNAi
causes phenotypes different from corresponding RNAi experiments in
Drosophila
and
bicoid
mutant embryos.
Megaselia-bicoid
is required not only for the head
and thorax but also for the development of four abdominal segments.
This difference between
Megaselia
and
Drosophila
suggests that the range of functional
bicoid
activity has been reduced in higher flies.
Combinatorial roles for pRB, p107, and p130 in E2F-mediated cell cycle control
Authors: Classon Marie , Salama Sofie , Gorka Carolyn , Mulloy Roseann , Braun Pascal , Harlow Ed
Published: July 2002

Numerous studies have implicated the pRB family of nuclear proteins
in the control of cell cycle progression. Although over-expression
experiments have revealed that each of these proteins, pRB, p107, and
p130, can induce a G
1
cell cycle arrest, mouse knockouts
demonstrated distinct developmental requirements for these proteins, as
well as partial functional redundancy between family members. To study
the mechanism by which the closely related pRB family proteins
contribute to cell cycle progression, we generated 3T3 fibroblasts
derived from embryos that lack one or more of these proteins
(pRB
−/−
, p107
−/−
, p130
−/−
,
pRB
−/−
/p107
−/−
,
pRB
−/−
/p130
−/−
, and
p107
−/−
/p130
−/−
). By comparing the
growth and cell cycle characteristics of these cells, we have observed
clear differences in the manner in which they transit through the
G
1
and S phases as well as exit from the cell cycle.
Deletion of
Rb
, or more than one of the family members,
results in a shortening of G
1
and a lengthening of S phase,
as well as a reduction in growth factor requirements. In addition, the
individual cell lines showed differential regulation of a subset of
E2F-dependent gene promoters, as well as differences in cell
cycle-dependent kinase activity. Taken together, these observations
suggest that the closely related pRB family proteins affect cell cycle
progression through distinct biochemical mechanisms and that their
coordinated action may contribute to their diverse functions in various
physiological settings.
Misregulation of gene expression in primary fibroblasts lacking poly(ADP-ribose) polymerase
Authors: Simbulan-Rosenthal Cynthia M. , Ly Danith H. , Rosenthal Dean S. , Konopka Genevieve , Luo RuiBai , Wang Zhao-Qi , Schultz Peter G. , Smulson Mark E.
Published: July 2002

Poly(ADP-ribose) polymerase (PARP) is implicated in the
maintenance of genomic integrity, given that inhibition or depletion of
this enzyme increases genomic instability in cells exposed to genotoxic
agents. We previously showed that immortalized fibroblasts derived from
PARP
−/−
mice exhibit an unstable tetraploid population,
and partial chromosomal gains and losses in PARP
−/−
mice
and immortalized fibroblasts are accompanied by changes in the
expression of p53, Rb, and c-Jun, as well as other proteins. A
tetraploid population has also now been detected in primary fibroblasts
derived from PARP
−/−
mice. Oligonucleotide microarray
analysis was applied to characterize more comprehensively the
differences in gene expression between asynchronously dividing primary
fibroblasts derived from PARP
−/−
mice and their wild-type
littermates. Of the 11,000 genes monitored, 91 differentially expressed
genes were identified. The loss of PARP results in down-regulation of
the expression of several genes involved in regulation of cell cycle
progression or mitosis, DNA replication, or chromosomal processing or
assembly. PARP deficiency also up-regulates genes that encode
extracellular matrix or cytoskeletal proteins that are implicated in
cancer initiation or progression or in normal or premature aging. These
results provide insight into the mechanism by which PARP deficiency
impairs mitotic function, thereby resulting in the genomic alterations
and chromosomal abnormalities as well as in altered expression of genes
that may contribute to genomic instability, cancer, and aging.
A conserved mechanism of retrovirus restriction in mammals
Authors: Towers Greg , Bock Michael , Martin Samia , Takeuchi Yasuhiro , Stoye Jonathan P. , Danos Olivier
Published: July 2002

The murine
Fv1
gene restricts infection by N- or
B-tropic murine leukemia viruses at a postentry, preintegration stage.
The
Fv1-
sensitive viruses previously used for the study
of
Fv1
encode an ecotropic envelope gene and thus only
infect rodent cells. Consequently, the study of
Fv1
restriction has been carried out solely in mice and murine cell lines.
By infection with retroviral vectors containing N- or B-tropic core and
pantropic vesicular stomatitis virus-G envelope protein, we now
demonstrate that cell lines derived from various mammalian species,
including humans, have an
Fv1
-like retrovirus
restriction function, preventing N-tropic vector infection. Like
Fv1
, restriction is directed at amino acid 110 of the
viral capsid protein. In contrast to
Fv1
, the novel
restriction is characterized by the absence of reverse-transcribed
viral DNA. We speculate that these activities have been selected for by
retroviral epidemics in the distant past.
Can coelenterates make coelenterazine? Dietary requirement for luciferin in cnidarian bioluminescence
Authors: Haddock Steven H. D. , Rivers Trevor J. , Robison Bruce H.
Published: July 2002

In the calcium-activated photoprotein aequorin, light is produced
by the oxidation of coelenterazine, the luciferin used by at least
seven marine phyla. However, despite extensive research on
photoproteins, there has been no evidence to indicate the origin of
coelenterazine within the phylum Cnidaria. Here we report that the
hydromedusa
Aequorea victoria
is unable to produce its
own coelenterazine and is dependent on a dietary supply of this
luciferin for bioluminescence. Although they contain functional
apophotoproteins, medusae reared on a luciferin-free diet are unable to
produce light unless provided with coelenterazine from an external
source. This evidence regarding the origins of luciferin in Cnidaria
has implications for the evolution of bioluminescence and for the
extensive use of coelenterazine among marine organisms.
Inhibition of cardiac L-type calcium channels by protein kinase C phosphorylation of two sites in the N-terminal domain
Authors: McHugh Damian , Sharp Elizabeth M. , Scheuer Todd , Catterall William A.
Published: July 2002

We have investigated the mechanism underlying the modulation of the
cardiac L-type Ca
2+
current by protein kinase C (PKC).
Using the patch-clamp technique, we found that PKC activation by
4-α-phorbol 12-myristate 13-acetate (PMA) or
rac
-1-oleyl-2-acetylglycerol (OAG) caused a substantial
reduction in Ba
2+
current through Ca
v
1.2
channels composed of α
1
1.2, β
1b
, and
α
2
δ
1
subunits expressed in tsA-201 cells.
In contrast, Ba
2+
current through a cloned brain isoform of
the Ca
v
1.2 channel (rbC-II) was unaffected by PKC
activation. Two potential sites of PKC phosphorylation are present at
positions 27 and 31 in the cardiac form of Ca
v
1.2, but not
in the brain form. Deletion of N-terminal residues 2–46 prevented PKC
inhibition. Conversion of the threonines at positions 27 and 31 to
alanine also abolished the PKC sensitivity of Ca
v
1.2.
Mutant Ca
v
1.2 channels in which the threonines were
converted singly to alanines were also insensitive to PKC modulation,
suggesting that phosphorylation of both residues is required for
PKC-dependent modulation. Consistent with this, mutating each of the
threonines individually to aspartate in separate mutants restored the
PKC sensitivity of Ca
v
1.2, indicating that a change in net
charge by phosphorylation of both sites is responsible for inhibition.
Our results define the molecular basis for inhibition of cardiac
Ca
v
1.2 channels by the PKC pathway.
SNARE-complex disassembly by NSF follows synaptic-vesicle fusion
Authors: Littleton J. Troy , Barnard Richard J. O. , Titus Steven A. , Slind Jessica , Chapman Edwin R. , Ganetzky Barry
Published: July 2002

Soluble
N-
ethylmaleimide-sensitive fusion attachment protein receptor (SNARE)-mediated fusion of synaptic vesicles with the presynaptic-plasma membrane is essential for communication between neurons. Disassembly of the SNARE complex requires the ATPase
N
-ethylmaleimide-sensitive fusion protein (NSF). To determine where in the synaptic-vesicle cycle NSF functions, we have undertaken a genetic analysis of
comatose
(dNSF-1) in
Drosophila
. Characterization of 16
comatose
mutations demonstrates that NSF mediates disassembly of SNARE complexes after synaptic-vesicle fusion. Hypomorphic mutations in NSF cause temperature-sensitive paralysis, whereas null mutations result in lethality. Genetic-interaction studies with
para
demonstrate that blocking evoked fusion delays the accumulation of assembled SNARE complexes and behavioral paralysis that normally occurs in
comatose
mutants, indicating NSF activity is not required in the absence of vesicle fusion. In addition, the entire vesicle pool can be depleted in
shibire comatose
double mutants, demonstrating that NSF activity is not required for the fusion step itself. Multiple rounds of vesicle fusion in the absence of NSF activity poisons neurotransmission by trapping SNAREs into cis-complexes. These data indicate that NSF normally dissociates and recycles SNARE proteins during the interval between exocytosis and endocytosis. In the absence of NSF activity, there are sufficient fusion-competent SNAREs to exocytose both the readily released and the reserve pool of synaptic vesicles.
“Kiss and run” exocytosis at hippocampal synapses
Authors: Stevens Charles F. , Williams James H.
Published: July 2002

We have combined electrophysiology and imaging to measure the
release of neurotransmitter and fluorescent dye at synapses of cultured
hippocampal neurons. These experiments have revealed a “kiss and
run” mode of exocytosis in which synaptic vesicles release glutamate
normally but do not permit dye to enter or escape from the vesicle.
During “kiss and run,” the vesicle interior may be exposed very
transiently (<6 ms), or a special configuration of the fusion pore may
prevent dye exchange. We estimate that about 20% of the vesicles
normally use this “kiss and run” pathway, and that the fraction
of “kiss and run” events can be increased to over 80% by
superfusing the synapses with hypertonic solution.
Terminal-repeat retrotransposons in miniature (TRIM) are involved in restructuring plant genomes
Authors: Witte Claus-Peter , Le Quang Hien , Bureau Thomas , Kumar Amar
Published: July 2002

A new group of long terminal repeats (LTR) retrotransposons, termed
t
erminal-
r
epeat retrotransposons
i
n
m
iniature (TRIM), are described that are present in both monocotyledonous and dicotyledonous plant. TRIM elements have terminal direct repeat sequences between ≈100 and 250 bp in length that encompass an internal domain of ≈100–300 bp. The internal domain contains primer binding site and polypurine tract motifs but lacks the coding domains required for mobility. Thus TRIM elements are not capable of autonomous transposition and probably require the help of mobility-related proteins encoded by other retrotransposons. The structural organization of TRIM elements suggests an evolutionary relationship to either LTR retrotransposons or retroviruses. The past mobility of TRIM elements is indicated by the presence of flanking 5-bp direct repeats found typically at LTR retrotransposon insertion sites, the high degree of sequence conservation between elements from different genomic locations, and the identification of
r
elated to
e
mpty
sites
(RESites). TRIM elements seem to be involved actively in the restructuring of plant genomes, affecting the promoter, coding region and intron-exon structure of genes. In solanaceous species and maize, TRIM elements provided target sites for further retrotransposon insertions. In
Arabidopsis
, evidence is provided that the TRIM element also can be involved in the transduction of host genes.
The pattern of gene expression in human CD34
+
stem/progenitor cells
Authors: Zhou Guolin , Chen Jianjun , Lee Sanggyu , Clark Terry , Rowley Janet D. , Wang San Ming
Published: July 2002

We have analyzed the pattern of gene expression in human primary CD34
+
stem/progenitor cells. We identified 42,399 unique serial analysis of gene expression (SAGE) tags among 106,021 SAGE tags collected from 2.5 × 10
6
CD34
+
cells purified from bone marrow. Of these unique SAGE tags, 21,546 matched known expressed sequences, including 3,687 known genes, and 20,854 were novel without a match. The SAGE tags that matched known sequences tended to be at higher levels, whereas the novel SAGE tags tended to be at lower levels. By using the generation of longer sequences from SAGE tags for gene identification (GLGI) method, we identified the correct gene for 385 of 440 high-copy SAGE tags that matched multiple genes and we generated 198 novel 3′ expressed sequence tags from 138 high-copy novel SAGE tags. We observed that many different SAGE tags were derived from the same genes, reflecting the high heterogeneity of the 3′ untranslated region in the expressed genes. We compared the quantitative relationship for genes known to be important in hematopoiesis. The qualitative identification and quantitative measure for each known gene, expressed sequence tag, and novel SAGE tag provide a base for studying normal gene expression in hematopoietic stem/progenitor cells and for studying abnormal gene expression in hematopoietic diseases.
MHC I-dependent antigen presentation is inhibited by poliovirus protein 3A
Authors: Deitz Stephen B. , Dodd Dana A. , Cooper Stewart , Parham Peter , Kirkegaard Karla
Published: July 2002

The effects of poliovirus 3A protein expression and poliovirus infection on the presentation of hepatitis C virus antigens in cultured chimpanzee cells were examined. Expression of poliovirus 3A protein inhibits protein secretion when expressed in isolation and was sufficient to protect chimpanzee cells from lysis by hepatitis C virus-specific cytotoxic T cells in standard51Cr-release assays. Poliovirus infection also inhibited antigen presentation, as determined by decreased cytotoxic T cell activation. A mutation in 3A that abrogates the inhibition of protein secretion also abolished the effects of poliovirus on antigen presentation. These results demonstrate that the inhibition of secretion observed in poliovirus-infected cells substantially reduces the presentation of new antigens on the cell surface. These observations may reflect a general mechanism by which nonenveloped viruses such as poliovirus and other viruses that do not require a functional protein secretory apparatus can evade detection by the cellular immune response.
The nature of fluorescence emission in the red fluorescent protein DsRed, revealed by single-molecule detection
Authors: Garcia-Parajo M. F. , Koopman M. , van Dijk E. M. H. P. , Subramaniam V. , van Hulst N. F.
Published: July 2002

Recent studies on the newly cloned red fluorescence
protein DsRed from the
Discosoma
genus have shown its
tremendous advantages: bright red fluorescence and high resistance
against photobleaching. However, it has also become clear that the
protein forms closely packed tetramers, and there is indication for
incomplete protein maturation with unknown proportion of immature green
species. We have applied single-molecule methodology to elucidate the
nature of the fluorescence emission in the DsRed. Real-time
fluorescence trajectories have been acquired with polarization
sensitive detection. Our results indicate that energy transfer between
identical monomers occurs efficiently with red emission arising equally
likely from any of the chromophoric units. Photodissociation of one of
the chromophores weakly quenches the emission of adjacent ones. Dual
color excitation (at 488 and 568 nm) single-molecule microscopy has
been performed to reveal the number and distribution of red vs. green
species within each tetramer. We find that 86% of the DsRed contain at
least one green species with a red-to-green ratio of 1.2–1.5. On the
basis of our findings, oligomer suppression would not only be
advantageous for protein fusion but will also increase the fluorescence
emission of individual monomers.
Induction of the WAF1/CIP1 protein and apoptosis in human T-cell leukemia virus type I-transformed lymphocytes after treatment with adriamycin by using a p53-independent pathway.
Authors: Gartenhaus R B , Wang P , Hoffmann P
Published: July 2002

The WAF1/CIP1 protein has been identified as a downstream mediator of the tumor suppressor p53 in regulating cell cycle progression through a G1-phase check-point. Recent work has implicated the functional status of p53 as a critical determinant in the apoptotic response of certain cell lines to DNA damaging agents. By using human T-cell leukemia virus type I-transformed lymphoid cell lines that differ in their level and function of wild-type p53, we investigated the induction of WAF1/CIP1 and apoptosis after exposure to Adriamycin, a genotoxic agent. We found that regardless of the p53 status in these cell lines, WAF1/CIP1 RNA was rapidly induced in response to Adriamycin treatment. An elevated level of WAF1/CIP1 protein was observed as well. Additionally, we demonstrated that apoptosis was induced in all cell lines analyzed despite some having functionally inactive p53 protein. Our data suggest that a p53-independent pathway may play a role in the apoptotic response observed in some cell lines after exposure to DNA damaging agents.
Controlling protein association and subcellular localization with a synthetic ligand that induces heterodimerization of proteins.
Authors: Belshaw P J , Ho S N , Crabtree G R , Schreiber S L
Published: July 2002

Extracellular growth and differentiation factors induce changes in gene expression in the nucleus by initiating a series of protein associations that alter the subcellular localization of intracellular signaling proteins. Initial events involve receptor homo- or heterodimerization and subsequent recruitment of cytosolic signaling proteins to the inner leaflet of the plasma membrane. Intermediate events involve the translocation of proteins into the nucleus. Late events involve the recruitment of transcriptional activators to the vicinity of specific genes in the nucleus, resulting in increased gene transcription. The ability to induce signals at each of these three phases of signaling pathways is illustrated by the use of a heterodimeric chemical inducer of dimerization that causes a proximal relationship between two different target proteins.
Late Holocene human-induced modifications to a central Polynesian island ecosystem.
Authors: Kirch P V
Published: July 2002

A 7000-year-long sequence of environmental change during the Holocene has been reconstructed for a central Pacific island (Mangaia, Cook Islands). The research design used geomorphological and palynological methods to reconstruct vegetation history, fire regime, and erosion and depositional rates, whereas archaeological methods were used to determine prehistoric Polynesian land use and resource exploitation. Certain mid-Holocene environmental changes are putatively linked with natural phenomena such as eustatic sea-level rise and periodic El Niño-Southern Oscillation events. However, the most significant changes were initiated between 2500 and 1800 years and were directly or indirectly associated with colonization by seafaring Polynesian peoples. These human-induced effects included major forest clearance, increased erosion of volcanic hillsides and alluvial deposition in valley bottoms, significant increases in charcoal influx, extinctions of endemic terrestrial species, and the introduction of exotic species.
Insights into antibody catalysis: structure of an oxygenation catalyst at 1.9-angstrom resolution.
Authors: Hsieh-Wilson L C , Schultz P G , Stevens R C
Published: July 2002

The x-ray crystal structures of the sulfide oxidase antibody 28B4 and of antibody 28B4 complexed with hapten have been solved at 2.2-angstrom and 1.9-angstrom resolution, respectively. To our knowledge, these structures are the highest resolution catalytic antibody structures to date and provide insight into the molecular mechanism of this antibody-catalyzed monooxygenation reaction. Specifically, the data suggest that entropic restriction plays a fundamental role in catalysis through the precise alignment of the thioether substrate and oxidant. The antibody active site also stabilizes developing charge on both sulfur and periodate in the transition state via cation-pi and electrostatic interactions, respectively. In addition to demonstrating that the active site of antibody 28B4 does indeed reflect the mechanistic information programmed in the aminophosphonic acid hapten, these high-resolution structures provide a basis for enhancing turnover rates through mutagenesis and improved hapten design.
Cystic fibrosis gene encodes a cAMP-dependent chloride channel in heart.
Authors: Hart P , Warth J D , Levesque P C , Collier M L , Geary Y , Horowitz B , Hume J R
Published: July 2002

cAMP-dependent chloride channels in heart contribute to autonomic regulation of action potential duration and membrane potential and have been inferred to be due to cardiac expression of the epithelial cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel. In this report, a cDNA from rabbit ventricle was isolated and sequenced, which encodes an exon 5 splice variant (exon 5-) of CFTR, with >90% identity to human CFTR cDNA present in epithelial cells. Expression of this cDNA in Xenopus oocytes gave rise to robust cAMP-activated chloride currents that were absent in control water-injected oocytes. Antisense oligodeoxynucleotides directed against CFTR significantly reduced the density of cAMP-dependent chloride currents in acutely cultured myocytes, thereby establishing a direct functional link between cardiac expression of CFTR protein and an endogenous chloride channel in native cardiac myocytes.
Antifreeze glycoproteins inhibit leakage from liposomes during thermotropic phase transitions.
Authors: Hays L M , Feeney R E , Crowe L M , Crowe J H , Oliver A E
Published: July 2002

Antifreeze glycoproteins (AFGPs), found in the blood of polar fish at concentrations as high as 35 g/liter, are known to prevent ice crystal growth and depress the freezing temperature of the blood. Previously, Rubinsky et al. [Rubinsky, B., Mattioli, M., Arav, A., Barboni, B. & Fletcher, G. L. (1992) Am. J. Physiol. 262, R542-R545] provided evidence that AFGPs block ion fluxes across membranes during cooling, an effect that they ascribed to interactions with ion channels. We investigated the effects of AFGPs on the leakage of a trapped marker from liposomes during chilling. As these liposomes are cooled through the transition temperature, they leak approximately 50% of their contents. Addition of less than 1 mg/ml of AFGP prevents up to 100% of this leakage, both during chilling and warming through the phase transition. This is a general effect that we show here applies to liposomes composed of phospholipids with transition temperatures ranging from 12 degrees C to 41 degrees C. Because these results were obtained with liposomes composed of phospholipids alone, we conclude that the stabilizing effects of AFGPs on intact cells during chilling reported by Rubinsky et al. may be due to a nonspecific effect on the lipid components of native membranes. There are other proteins that prevent leakage, but only under specialized conditions. For instance, antifreeze proteins, bovine serum albumin, and ovomucoid all either have no effect or actually induce leakage. Following precipitation with acetone, all three proteins inhibited leakage, although not to the extent seen with AFGPs. Alternatively, there are proteins such as ovotransferrin that have no effect on leakage, either before or after acetone precipitation.
Direct measurement of oligonucleotide binding stoichiometry of gene V protein by mass spectrometry.
Authors: Cheng X , Harms A C , Goudreau P N , Terwilliger T C , Smith R D
Published: July 2002

The binding stoichiometry of gene V protein from bacteriophage f1 to several oligonucleotides was studied using electrospray ionization-mass spectrometry (ESI-MS). Using mild mass spectrometer interface conditions that preserve noncovalent associations in solution, gene V protein was observed as dimer ions from a 10 mM NH4OAc solution. Addition of oligonucleotides resulted in formation of protein-oligonucleotide complexes with stoichiometry of approximately four nucleotides (nt) per protein monomer. A 16-mer oligonucleotide gave predominantly a 4:1 (protein monomer: oligonucleotide) complex while oligonucleotides shorter than 15 nt showed stoichiometries of 2:1. Stoichiometries and relative binding constants for a mixture of oligonucleotides were readily measured using mass spectrometry. The binding stoichiometry of the protein with the 16-mer oligonucleotide was measured independently using size-exclusion chromatography and the results were consistent with the mass spectrometric data. These results demonstrate, for the first time, the observation and stoichiometric measurement of protein-oligonucleotide complexes using ESI-MS. The sensitivity and high resolution of ESI-MS should make it a useful too] in the study of protein-DNA interactions.
Mechanism of anti-HIV action of masked alaninyl d4T-MP derivatives.
Authors: Balzarini J , Karlsson A , Aquaro S , Perno C F , Cahard D , Naesens L , De Clercq E , McGuigan C
Published: July 2002

So324 is a 2',3'-dideoxy-2',3'-didehydrothymidine-5'-monophosphate (d4T-MP) prodrug containing at the phosphate moiety a phenyl group and the methylester of alanine linked to the phosphate through a phosphoramidate linkage. So324 has anti-HIV activity in human CEM, MT4, and monocyte/macrophage cells that is superior to that of d4T. In contrast to d4T, So324 is also able to inhibit HIV replication in thymidine kinase-deficient CEM cells. After uptake of So324 by intact human lymphocytes, d4T-MP is released and subsequently converted intracellularly to d4T-TP. In addition, accumulation of substantial amounts of a novel d4T derivative has been found. This d4T metabolite has been characterized as alaninyl d4T-MP. The latter metabolite accumulates at approximately 13- to 200-fold higher levels than d4T-TP depending the experimental conditions. Alaninyl d4T-MP should be considered as an intra- and/or extracellular depot form of d4T and/or d4T-MP. These findings may explain the superior anti-retroviral activity of So324 over d4T in cell culture.