bluuebunny/crossref_metadata_2025 huggingface dataset:
Apoptosis of nur77/N10-transgenic thymocytes involves the Fas/Fas ligand pathway.
Authors: Weih F , Ryseck R P , Chen L , Bravo R
Published: July 2002

The orphan nuclear receptor Nur77/N10 has recently been demonstrated to be involved in apoptosis of T cell hybridomas. We report here that chronic expression of Nur77/N10 in thymocytes of transgenic mice results in a dramatic reduction of CD4+CD8+ double-positive as well as CD4+CD8- and CD4-CD8+ single-positive cell populations due to an early onset of apoptosis. CD4-CD8- double-negative and CD25+ precursor cells, however, are unaffected. Moreover, nur77/N10-transgenic thymocytes show increased expression of Fas ligand (FasL), while the levels of the Fas receptor (Fas) are not increased. The mouse spontaneous mutant gld (generalized lymphoproliferative disease) carries a point mutation in the extracellular domain of the FasL gene that abolishes the ability of FasL to bind to Fas. Thymuses from nur77/N10-transgenic mice on a gld/gld background have increased cellularity and an almost normal profile of thymocyte subpopulations. Our results demonstrate that one pathway of apoptosis triggered by Nur77/N10 in double-positive thymocytes occurs through the upregulation of FasL expression resulting in increased signaling through Fas.
ECA39, a conserved gene regulated by c-Myc in mice, is involved in G1/S cell cycle regulation in yeast.
Authors: Schuldiner O , Eden A , Ben-Yosef T , Yanuka O , Simchen G , Benvenisty N
Published: July 2002

The c-myc oncogene has been shown to play a role in cell proliferation and apoptosis. The realization that myc oncogenes may control the level of expression of other genes has opened the field to search for genetic targets for Myc regulation. Recently, using a subtraction/coexpression strategy, a murine genetic target for Myc regulation, called EC439, was isolated. To further characterize the ECA39 gene, we set out to determine the evolutionary conservation of its regulatory and coding sequences. We describe the human, nematode, and budding yeast homologs of the mouse ECA39 gene. Identities between the mouse ECA39 protein and the human, nematode, or yeast proteins are 79%, 52%, and 49%, respectively. Interestingly, the recognition site for Myc binding, located 3' to the start site of transcription in the mouse gene, is also conserved in the human homolog. This regulatory element is missing in the ECA39 homologs from nematode or yeast, which also lack the regulator c-myc. To understand the function of ECA39, we deleted the gene from the yeast genome. Disruption of ECA39 which is a recessive mutation that leads to a marked alteration in the cell cycle. Mutant haploids and homozygous diploids have a faster growth rate than isogenic wild-type strains. Fluorescence-activated cell sorter analyses indicate that the mutation shortens the G1 stage in the cell cycle. Moreover, mutant strains show higher rates of UV-induced mutations. The results suggest that the product of ECA39 is involved in the regulation of G1 to S transition.
DP-2, a heterodimeric partner of E2F: identification and characterization of DP-2 proteins expressed in vivo.
Authors: Rogers K T , Higgins P D , Milla M M , Phillips R S , Horowitz J M
Published: July 2002

E2F is a heterodimeric transcription factor that regulates the expression of genes at the G1/S boundary and is composed of two related but distinct families of proteins, E2F and DP. E2F/DP heterodimers form complexes with the retinoblastoma (Rb) protein, the Rb-related proteins p107 and p130, and cyclins/cdks in a cell cycle-dependent fashion in vivo. E2F is encoded by at least five closely related genes, E2F-1 through -5. Here we report studies of DP-2, the second member of the DP family of genes. Our results indicate that (i) DP-2 encodes at least five distinct mRNAs, (ii) a site of alternative splicing occurs within the 5' untranslated region of DP-2 mRNA, (iii) at least three DP-2-related proteins (of 55, 48, and 43 kDa) are expressed in vivo, (iv) each of these proteins is phosphorylated, and (v) one DP-2 protein (43 kDa) carries a truncated amino terminus. Our data also strongly suggest that the 55-kDa DP-2-related protein is a novel DP-2 isoform that results from alternative splicing. Thus, we conclude that DP-2 encodes a set of structurally, and perhaps functionally, distinct proteins in vivo.
The genesis of avian neural crest cells: a classic embryonic induction.
Authors: Selleck M A , Bronner-Fraser M
Published: July 2002

Neural crest cells arise from the ectoderm and are first recognizable as discrete cells in the chicken embryo when they emerge from the neural tube. Despite the classical view that neural crest precursors are a distinct population lying between epidermis and neuroepithelium, our results demonstrate that they are not a segregated population. Cell lineage analyses have demonstrated that individual precursor cells within the neural folds can give rise to epidermal, neural crest, and neural tube derivatives. Interactions between the neural plate and epidermis can generate neural crest cells, since juxtaposition of these tissues at early stages results in the formation of neural crest cells at the interface. Inductive interactions between the epidermis and neural plate can also result in "dorsalization" of the neural plate, as assayed by the expression of the Wnt transcripts characteristic of the dorsal neural tube. The competence of the neural plate changes with time, however, such that interaction of early neural plate with epidermis generates only neural crest cells, whereas interaction of slightly older neural plate with epidermis generates neural crest cells and Wnt-expressing cells. At cranial levels, neuroepithelial cells can regulate to generate neural crest cells when the endogenous neural folds are removed, probably via interaction of the remaining neural tube with the epidermis. Taken together, these experiments demonstrate that: (i) progenitor cells in the neural folds are multipotent, having the ability to form multiple ectodermal derivatives, including epidermal, neural crest, and neural tube cells; (ii) the neural crest is an induced population that arises by interactions between the neural plate and the epidermis; and (iii) the competence of the neural plate to respond to inductive interactions changes as a function of embryonic age.
CD30/TNF receptor-associated factor interaction: NF-kappa B activation and binding specificity.
Authors: Lee S Y , Lee S Y , Kandala G , Liou M L , Liou H C , Choi Y
Published: July 2002

CD30 is a member of the tumor necrosis factor (TNF) receptor superfamily. CD30 is expressed on normal activated lymphocytes, on several virally transformed T- or B-cell lines and on neoplastic cells of Hodgkin's lymphoma. The interaction of CD30 with its ligand induces pleiotropic effects on cells resulting in proliferation, differentiation, or death. The CD30 cytoplasmic tail interacts with TNF receptor-associated factors (TRAFs), which have been shown to transduce signals mediated by TNF-R2 and CD40. We demonstrate here that TRAF2 also plays an important role in CD30-induced NF-kappa B activation. We also show that TRAF2-mediated activation of NF-kappa B plays a role in the activation of HIV transcription induced by CD30 cross-linking. Detailed site-directed mutagenesis of the CD30 cytoplasmic tail reveals that there are two independent binding sites for TRAF, each interacting with a different domain of TRAF. Furthermore, we localized the TRAF-C binding site in CD30 to a 5-7 amino acid stretch.
Parallel human genome analysis: microarray-based expression monitoring of 1000 genes.
Authors: Schena M , Shalon D , Heller R , Chai A , Brown P O , Davis R W
Published: July 2002

Microarrays containing 1046 human cDNAs of unknown sequence were printed on glass with high-speed robotics. These 1.0-cm2 DNA "chips" were used to quantitatively monitor differential expression of the cognate human genes using a highly sensitive two-color hybridization assay. Array elements that displayed differential expression patterns under given experimental conditions were characterized by sequencing. The identification of known and novel heat shock and phorbol ester-regulated genes in human T cells demonstrates the sensitivity of the assay. Parallel gene analysis with microarrays provides a rapid and efficient method for large-scale human gene discovery.
Activation of an interleukin 1 converting enzyme-dependent apoptosis pathway by granzyme B.
Authors: Shi L , Chen G , MacDonald G , Bergeron L , Li H , Miura M , Rotello R J , Miller D K , Li P , Seshadri T , Yuan J , Greenberg A H
Published: July 2002

Cytotoxic T lymphocytes (CTL) can induce apoptosis through a granzyme B-based killing mechanism. Here we show that in cells undergoing apoptosis by granzyme B, both p45 pro-interleukin 1 beta converting enzyme (ICE) and pro-CPP32 are processed. Using ICE deficient (ICE -/-) mice, embryonic fibroblasts exhibit high levels of resistance to apoptosis by granzyme B or granzyme 3, while B lymphoblasts are granzyme B-resistant, thus identifying an ICE-dependent apoptotic pathway that is activated by CTL granzymes. In contrast, an alternative ICE-independent pathway must also be activated as ICE -/- thymocytes remain susceptible to apoptosis by both granzymes. In ICE -/- B cells or HeLa cells transfected with mutant inactive ICE or Ich-1S that exhibit resistance to granzyme B, CPP32 is processed to p17 and poly(ADP-ribose) polymerase is cleaved indicating that this protease although activated was not associated with an apoptotic nuclear phenotype. Using the peptide inhibitor Ac-DEVD-CHO, apoptosis as well as p45 ICE hydrolysis are suppressed in HeLa cells, suggesting that a CPP32-like protease is upstream of ICE. In contrast, p34cdc2 kinase, which is required for granzyme B-induced apoptosis, remains inactive in ICE -/- B cells indicating it is downstream of ICE. We conclude that granzyme B activates an ICE-dependent cell death pathway in some cell types and requires a CPP32-like Ac-DEVD-CHO inhibitable protease acting upstream to initiate apoptosis.
RNA replication by Q beta replicase: a working model.
Authors: Brown D , Gold L
Published: July 2002

Two classes of RNA ligands that bound to separate, high affinity nucleic acid binding sites on Q beta replicase were previously identified. RNA ligands to the two sites, referred to as site I and site II, were used to investigate the molecular mechanism of RNA replication employed by the four-subunit replicase. Replication inhibition by site I- and site II-specific ligands defined two subsets of replicatable RNAs. When provided with appropriate 3' ends, ligands to either site served as replication templates. UV crosslinking experiments revealed that site I is associated with the S1 subunit, site II with elongation factor Tu, and polymerization with the viral subunit of the holoenzyme. These results provide the framework for a three site model describing template recognition and product strand initiation by Q beta replicase.
The chemistry of the
S
-nitrosoglutathione/glutathione system
Authors: Singh S. P. , Wishnok J. S. , Keshive M. , Deen W. M. , Tannenbaum S. R.
Published: July 2002

S
-Nitrosothiols have generated considerable interest
due to their ability to act as nitric oxide (NO) donors and due to
their possible involvement in bioregulatory systems—e.g., NO transfer
reactions. Elucidation of the reaction pathways involved in the
modification of the thiol group by
S
-nitrosothiols is
important for understanding the role of
S
-nitroso compounds
in vivo
. The modification of glutathione (GSH) in the
presence of
S
-nitrosoglutathione (GSNO) was examined as a
model reaction. Incubation of GSNO (1 mM) with GSH at various
concentrations (1–10 mM) in phosphate buffer (pH 7.4) yielded oxidized
glutathione, nitrite, nitrous oxide, and ammonia as end products. The
product yields were dependent on the concentrations of GSH and oxygen.
Transient signals corresponding to GSH conjugates, which increased by
one mass unit when the reaction was carried out with
15
N-labeled GSNO, were identified by electrospray
ionization mass spectrometry. When morpholine was present in the
reaction system,
N
-nitrosomorpholine was formed. Increasing
concentrations of either phosphate or GSH led to lower yields of
N
-nitrosomorpholine. The inhibitory effect of phosphate may
be due to reaction with the nitrosating agent, nitrous anhydride
(N
2
O
3
), formed by oxidation of NO. This
supports the release of NO during the reaction of GSNO with GSH. The
products noted above account quantitatively for virtually all of the
GSNO nitrogen consumed during the reaction, and it is now possible to
construct a complete set of pathways for the complex transformations
arising from GSNO + GSH.
Oncogenic mutation in the Kit receptor tyrosine kinase alters substrate specificity and induces degradation of the protein tyrosine phosphatase SHP-1
Authors: Piao Xianhua , Paulson Robert , van der Geer Peter , Pawson Tony , Bernstein Alan
Published: July 2002

Activating mutations in the Kit receptor tyrosine kinase have been identified in both rodent and human mast cell leukemia. One activating Kit mutation substitutes a valine for aspartic acid at codon 816 (D816V) and is frequently observed in human mastocytosis. Mutation at the equivalent position in the murine c-kitgene, involving a substitution of tyrosine for aspartic acid (D814Y), has been described in the mouse mastocytoma cell line P815. We have investigated the mechanism of oncogenic activation by this mutation. Expression of this mutant Kit receptor tyrosine kinase in a mast cell line led to the selective tyrosine phosphorylation of a 130-kDa protein and the degradation, through the ubiquitin-dependent proteolytic pathway, of a 65-kDa phosphoprotein. The 65-kDa protein was identified as the src homology domain 2 (SH2)-containing protein tyrosine phosphatase SHP-1, a negative regulator of signaling by Kit and other hematopoietic receptors, and the protein product of the murine motheaten locus. This mutation also altered the sites of receptor autophosphorylation and peptide substrate selectivity. Thus, this mutation activates the oncogenic potential of Kit by a novel mechanism involving an alteration in Kit substrate recognition and the degradation of SHP-1, an attenuator of the Kit signaling pathway.
Identification of fission yeast nuclear markers using random polypeptide fusions with green fluorescent protein
Authors: Sawin Kenneth E. , Nurse Paul
Published: July 2002

We describe a method for identifying genes encoding proteins with
stereospecific intracellular localizations in the fission yeast
Schizosaccharomyces pombe
. Yeast are transformed with a
gene library in which
S. pombe
genomic sequences are
fused to the gene encoding the
Aequorea victoria
green
fluorescent protein (GFP), and intracellular localizations are
subsequently identified by rapid fluorescence screening
in
vivo
. In a model application of these methods to the fission
yeast nucleus, we have identified several novel genes whose products
are found in specific nuclear regions, including chromatin, the
nucleolus, and the mitotic spindle, and sequence similarities between
some of these genes and previously identified genes encoding nuclear
proteins have validated the approach. These methods will be useful in
identifying additional components of the
S. pombe
nucleus, and further extensions of this approach should also be
applicable to a more comprehensive identification of the elements of
intracellular architecture in fission yeast.
Rescue of a segmented negative-strand RNA virus entirely from cloned complementary DNAs
Authors: Bridgen Anne , Elliott Richard M.
Published: July 2002

We provide the first report, to our knowledge, of a
helper-independent system for rescuing a segmented, negative-strand RNA
genome virus entirely from cloned cDNAs. Plasmids were constructed
containing full-length cDNA copies of the three Bunyamwera bunyavirus
RNA genome segments flanked by bacteriophage T7 promoter and hepatitis
delta virus ribozyme sequences. When cells expressing both
bacteriophage T7 RNA polymerase and recombinant Bunyamwera bunyavirus
proteins were transfected with these plasmids, full-length
antigenome RNAs were transcribed intracellularly, and these in turn
were replicated and packaged into infectious bunyavirus particles. The
resulting progeny virus contained specific genetic tags characteristic
of the parental cDNA clones. Reassortant viruses containing two genome
segments of Bunyamwera bunyavirus and one segment of Maguari bunyavirus
were also produced following transfection of appropriate plasmids. This
accomplishment will allow the full application of recombinant DNA
technology to manipulate the bunyavirus genome.
Helicobacter pylori attachment to gastric cells induces cytoskeletal rearrangements and tyrosine phosphorylation of host cell proteins.
Authors: Segal E D , Falkow S , Tompkins L S
Published: July 2002

The consequences of Helicobacter pylori attachment to human gastric cells were examined by transmission electron microscopy and immunofluorescence microscopy. H. pylori attachment resulted in (i) effacement of microvilli at the site of attachment, (ii) cytoskeletal rearrangement directly beneath the bacterium, and (iii) cup/pedestal formation at the site of attachment. Double-immunofluorescence studies revealed that the cytoskeletal components actin, alpha-actinin, and talin are involved in the process. Immunoblot analysis showed that binding of H. pylori to AGS cells induced tyrosine phosphorylation of two host cell proteins of 145 and 105 kDa. These results indicate that attachment of H. pylori to gastric epithelial cells resembles that of enteropathogenic Escherichia coli. Coccoid H. pylori, which are thought to be terminally differentiated bacterial forms, are capable of binding and inducing cellular changes of the same sort as spiral H. pylori, including tyrosine phosphorylation of host proteins.
Reaction of the Escherichia coli quinol oxidase cytochrome bo3 with dioxygen: the role of a bound ubiquinone molecule.
Authors: Puustinen A , Verkhovsky M I , Morgan J E , Belevich N P , Wikstrom M
Published: July 2002

We have studied the kinetics of the oxygen reaction of the fully reduced quinol oxidase, cytochrome bo3, using flow-flash and stopped flow techniques. This enzyme belongs to the heme-copper oxidase family but lacks the CuA center of the cytochrome c oxidases. Depending on the isolation procedure, the kinetics are found to be either nearly monophasic and very different from those of cytochrome c oxidase or multiphasic and quite similar to cytochrome c oxidase. The multiphasic kinetics in cytochrome c oxidase can largely be attributed to the presence Of CuA as the donor of a fourth electron, which rereduces the originally oxidized low-spin heme and completes the reduction of O2 to water. Monophasic kinetics would thus be expected, a priori, for cytochrome bo3 since it lacks the CuA center, and in this case we show that the oxygen reaction is incomplete and ends with the ferryl intermediate. Multiphasic kinetics thus suggest the presence of an extra electron donor (analogous to CuA). We observe such kinetics exclusively with cytochrome bo3 that contains a single equivalent of bound ubiquinone-8, whereas we find no bound ubiquinone in an enzyme exhibiting monophasic kinetics. Reconstitution with ubiquinone-8 converts the reaction kinetics from monophasic to multiphasic. We conclude that a single bound ubiquinone molecule in cytochrome bo3 is capable of fast rereduction of heme b and that the reaction with O2 is quite similar in quinol and cytochrome c oxidases.
Surface blebs on apoptotic cells are sites of enhanced procoagulant activity: implications for coagulation events and antigenic spread in systemic lupus erythematosus.
Authors: Casciola-Rosen L , Rosen A , Petri M , Schlissel M
Published: July 2002

The restriction of phosphatidylserine (PtdSer) to the inner surface of the plasma membrane bilayer is lost early during apoptosis. Since PtdSer is a potent surface procoagulant, and since there is an increased incidence of coagulation events in patients with systemic lupus erythematosus (SLE) who have anti-phospholipid antibodies, we addressed whether apoptotic cells are procoagulant and whether anti-phospholipid antibodies influence this. Apoptotic HeLa cells, human endothelial cells, and a murine pre-B-cell line were markedly procoagulant in a modified Russell viper venom assay. This procoagulant effect was entirely abolished by addition of the PtdSer-binding protein, annexin V, confirming that it was PtdSer-dependent. The procoagulant effect was also abolished by addition of IgG purified from the plasma of three patients with anti-phospholipid antibody syndrome, but not IgG from normal controls. Confocal microscopy of apoptotic cells stained with fluorescein-isothiocyanate-conjugated-annexin V demonstrated (Ca2+)-dependent binding to the surface of membrane blebs o apoptotic cells, but not to intracellular membranes. Recent data indicate that the surface blebs of apoptotic cells constitute an important immunogenic particle in SLE. We propose that the PtdSer exposed on the outside of these blebs can induce the production of anti-phospholipid antibodies, which might also enhance the immunogenicity of the bleb contents. When apoptosis occurs in a microenvironment in direct contact with circulating plasma, the unique procoagulant consequences of the apoptotic surface may additionally be expressed. This might explain the increased incidence of pathological intravascular coagulation events that occur in some lupus patients who have anti-phospholipid antibodies.
Repression of a matrix metalloprotease gene by E1A correlates with its ability to bind to cell type-specific transcription factor AP-2.
Authors: Somasundaram K , Jayaraman G , Williams T , Moran E , Frisch S , Thimmapaya B
Published: July 2002

Adenovirus E1A 243-amino acid protein can repress a variety of enhancer -linked viral and cellular promoters. This repression is presumed to be mediated by its interaction with and sequestration of p3OO, a transcriptional coactivator. Type IV 72-kDa collagenase is one of the matrix metalloproteases that has been implicated in differentiation, development, angiogenesis, and tumor metastasis. We show here that the cell type-specific transcription factor AP-2 is an important transcription factor for the activation of the type IV 72-kDa collagenase promoter and that adenovirus E1A 243-amino acid protein represses this promoter by targeting AP-2. Glutathione S-transferase-affinity chromatography studies show that the E1A protein interacts with the DNA binding/dimerization region of AP-2 and that the N-terminal amino acids of E1A protein are required for this interaction. Further, E1A deletion mutants which do not bind to p3OO can repress this collagenase promoter as efficiently as the wildtype E1A protein. Because the AP-2 element is present in a variety of viral and cellular enhancers which are repressed by E1A, these studies suggest that E1A protein can repress cellular and viral promoter/enhancers by forming a complex with cellular transcription factors and that this repression mechanism may be independent of its interaction with p3OO.
Resistance of actin to cleavage during apoptosis
Authors: Song Qizhong , Wei Tie , Lees-Miller Susan , Alnemri Emad , Watters Dianne , Lavin Martin F.
Published: July 2002

A small number of cellular proteins present in the nucleus, cytosol, and membrane fraction are specifically cleaved by the interleukin-1β-converting enzyme (ICE)-like family of proteases during apoptosis. Previous results have demonstrated that one of these, the cytoskeletal protein actin, is degraded in rat PC12 pheochromocytoma cells upon serum withdrawal. Extracts from etoposide-treated U937 cells are also capable of cleaving actin. It was assumed that cleavage of actin represented a general phenomenon, and a mechanism coordinating proteolytic, endonucleolytic, and morphological aspects of apoptosis was proposed. We demonstrate here that actin is resistant to degradation in several different human cells induced to undergo apoptosis in response to a variety of stimuli, including Fas ligation, serum withdrawal, cytotoxic T-cell killing, and DNA damage. On the other hand, cell-free extracts from these cells and the ICE-like protease CPP32 were capable of cleaving actinin vitro. We conclude that while actin contains cleavage sites for ICE-like proteases, it is not degradedin vivoin human cells either because of lack of access of these proteases to actin or due to the presence of other factors that prevent degradation.
Event-related brain potential correlates of two states of conscious awareness in memory
Authors: Düzel Emrah , Yonelinas Andrew P. , Mangun George R. , Heinze Hans-Jochen , Tulving Endel
Published: July 2002

We report an event-related potential (ERP) experiment of human recognition memory that explored the relation between conscious awareness and electrophysiological activity of the brain. We recorded ERPs from healthy adults while they made “remember” and “know” recognition judgments about previously seen words. These two kinds of judgments reflect “autonoetic” and “noetic” awareness, respectively. The ERP effects differed between the two kinds of awareness while they were similar for “true” and “false” recognition. Noetic awareness was associated with a temporoparietal positivity in the N400 range (325–600 ms) and a late (600–1,000 ms) frontocentral negativity, whereas autonoetic awareness was associated with a widespread, late, bifrontal and left parietotemporal (600–1000 ms) positivity. In the very late (1,300–1,900 ms) time window, a right frontal positivity was observed for both remember and know judgments of both true and false targets. These results provide physiological evidence for two types of conscious awareness in episodic memory retrieval.
Expression of a gene encoding a tRNA synthetase-like protein is enhanced in tumorigenic human myeloid leukemia cells and is cell cycle stage- and differentiation-dependent
Authors: Sen Subrata , Zhou Hongyi , Ripmaster Tracy , Hittelman Walter N. , Schimmel Paul , White R. Allen
Published: July 2002

We cloned a tumorigenic phenotype-associated cDNA encoding a tRNA synthetase-like protein from an acute-phase human myeloid leukemia cell line. The cDNA was isolated by reiterative subtraction of cDNAs synthesized from tumor-generating parental leukemia cells versus those from a nontumorigenic variant of the same cells. The selected cDNA encodes a protein that is a close homolog of one subunit of prokaryote and yeast phenylalanyl-tRNA synthetase (PheRS). The expressed protein reacts specificially with polyclonal antibodies raised against mammalian phenylalanyl-tRNA synthetase. Expression of the gene (designated CML33) was directly confirmed by Northern blot hybridization to be substantially enhanced in the tumorigenic cells compared with the nontumorigenic variant. In addition, expression of CML33 in myeloid leukemia cells was sensitive to the stage of the cell cycle and to induction of differentiation. Although the relationship between these observations and the tumorigenic state of the human myeloid leukemia cell line used in these studies is unknown, to our knowledge, this is the first demonstration in mammalian cells of tumor-selective and cell cycle stage- and differentiation-dependent expression of a member of the tRNA synthetase gene family.
Assembly and disassembly of a ternary complex of synaptobrevin, syntaxin, and SNAP-25 in the membrane of synaptic vesicles
Authors: Otto Henning , Hanson Phyllis I. , Jahn Reinhard
Published: July 2002

The synaptic membrane proteins synaptobrevin, syntaxin, and SNAP-25 form a ternary complex that can be disassembled by the ATPase
N
-ethylmaleimide-sensitive factor (NSF) in the presence of soluble cofactors (SNAP proteins). These steps are thought to represent molecular events involved in docking and subsequent exocytosis of synaptic vesicles. Using two independent and complementary approaches, we now report that such ternary complexes form in the membrane of highly purified and monodisperse synaptic vesicles in the absence of the plasma membrane. Furthermore, the complexes are reversibly dissociated by NSF and SNAP proteins. Thus, ternary complexes can be assembled and disassembled while all three proteins are anchored as neighbors in the same membrane, suggesting that NSF is involved in priming synaptic vesicles for exocytosis.
Cloning gibberellin dioxygenase genes from pumpkin endosperm by heterologous expression of enzyme activities in
Escherichia coli
Authors: Lange Theo
Published: July 2002

Gibberellin (GA) plant hormones are biosynthesized via complex pathways, the final steps of which are catalyzed by 2-oxoglutarate-dependent dioxygenases. Here, the cloning of two such enzymes, the GA 7-oxidase and the GA 20-oxidase, is reported using a novel approach, namely, by screening for GA dioxygenase activities expressed as T7 gene 10 fusion proteins in recombinant
Escherichia coli
.
In vitro
translation products of mRNA from endosperm of immature pumpkin seeds contained three GA dioxygenase activities, including 7-oxidase, 20-oxidase, and 3β-hydroxylase. A cDNA expression library was prepared from the endosperm mRNA in λMOS
Elox
. An aliquot of the amplified library was converted to plasmids
in vivo
and used for transformation of
E. coli
BL21(DE3), which thereafter expressed recombinant fusion proteins containing 7-oxidase, 20-oxidase, and 3β-hydroxylase activities. By screening for specific GA dioxygenase expression, clones harboring 7-oxidase and 20-oxidase cDNA were isolated. The ORF of the 7-oxidase cDNA is 945 bp long, encoding for 314 amino acid residues with a calculated
M
r
of 35,712 and pI of 5.7. Recombinant GA 7-oxidase oxidizes GA
12
-aldehyde to GA
12
and GA
14
-aldehyde to GA
14
. Evidence was obtained for further metabolism of GA
12
by the 7-oxidase to four products, two of which are monohydroxylated GA
12
. The ORF of the 20-oxidase is—apart from seven changes, resulting in four amino acid substitutions—identical to the 20-oxidase cDNA previously cloned from pumpkin cotyledon mRNA; both 20-oxidases have the same catalytic properties.
The AP2 domain of
APETALA2
defines a large new family of DNA binding proteins in
Arabidopsis
Authors: Okamuro Jack K. , Caster Brian , Villarroel Raimundo , Van Montagu Marc , Jofuku K. Diane
Published: July 2002

APETALA2
(
AP2
) plays an important role in
the control of Arabidopsis flower and seed development and encodes a
putative transcription factor that is distinguished by a novel DNA
binding motif referred to as the AP2 domain. In this study we show that
the AP2 domain containing or RAP2 (related to AP2) family of proteins
is encoded by a minimum of 12 genes in
Arabidopsis
. The
RAP2
genes encode two classes of proteins, AP2-like and
EREBP-like, that are defined by the number of AP2 domains in each
polypeptide as well as by two sequence motifs referred to as the YRG
and RAYD elements that are located within each AP2 domain.
RAP2
genes are differentially expressed in flower, leaf,
inflorescence stem, and root. Moreover, the expression of at least
three
RAP2
genes in vegetative tissues are controlled by
AP2
. Thus, unlike other floral homeotic genes,
AP2
is active during both reproductive and vegetative
development.
The mouse pale ear (
ep
) mutation is the homologue of human Hermansky–Pudlak syndrome
Authors: Gardner John M. , Wildenberg Scott C. , Keiper Natalie M. , Novak Edward K. , Rusiniak Michael E. , Swank Richard T. , Puri Neelu , Finger Joshua N. , Hagiwara Nobuko , Lehman Anne L. , Gales Tracy L. , Bayer Manfred E. , King Richard A. , Brilliant Murray H.
Published: July 2002

The recessive mutation at the pale ear (
ep
) locus on mouse chromosome 19 was found to be the homologue of human Hermansky–Pudlak syndrome (HPS). A positional cloning strategy using yeast artificial chromosomes spanning the HPS locus was used to identify the HPS gene and its murine counterpart. These genes and their predicted proteins are highly conserved at the nucleotide and amino acid levels. Sequence analysis of the mutant
ep
gene revealed the insertion of an intracisternal A particle element in a protein-coding 3′ exon. Here we demonstrate that mice with the
ep
mutation exhibit abnormalities similar to human HPS patients in melanosomes and platelet-dense granules. These results establish an animal model of HPS and will facilitate biochemical and molecular analyses of the functions of this protein in the membranes of specialized intracellular organelles.
Evidence of evolutionary up-regulation of the single active X chromosome in mammals based on
Clc
4 expression levels in
Mus spretus
and
Mus musculus
Authors: Adler David A. , Rugarli Elena I. , Lingenfelter Patricia A. , Tsuchiya Karen , Poslinski Diane , Liggitt H. Denny , Chapman Verne M. , Elliott Rosemary W. , Ballabio Andrea , Disteche Christine M.
Published: July 2002

Previous studies have shown that the chloride channel gene
Clc4
is X-linked and subject to X inactivation in
Mus spretus
, but that the same gene is autosomal in laboratory strains of mice. This exception to the conservation of linkage of the X chromosome in one of two interfertile mouse species was exploited to compare expression of
Clc4
from the X chromosome to that from the autosome.
Clc4
was found to be highly expressed in brain tissues of both mouse species. Quantitative analyses of species-specific expression of
Clc4
in brain tissues from mice resulting from
M. spretus
× laboratory strain crosses, demonstrate that each autosomal locus has half the level of
Clc4
expression as compared with the single active X-linked locus. In contrast expression of another chloride channel gene,
Clc3
, which is autosomal in both mouse species is equal between alleles in F
1
animals. There is no evidence of imprinting of the
Clc4
autosomal locus. These results are consistent with Ohno’s hypothesis of an evolutionary requirement for a higher expression of genes on the single active X chromosome to maintain balance with autosomal gene expression [Ohno, S. (1967)
Sex Chromosomes and Sex-Linked Genes
(Springer, Berlin)].
Lysis of HIV-1-infected cells and inhibition of viral replication by universal receptor T cells
Authors: Yang Otto O. , Tran Annie-Chen , Kalams Spyros A. , Johnson R. Paul , Roberts Margo R. , Walker Bruce D.
Published: July 2002

Increasing evidence suggests that HIV-1-specific cytotoxic T lymphocytes (CTLs) are a key host immune response to HIV-1 infection. Generation of CTL responses for prevention or therapy of HIV-1 infection has several intrinsic technical barriers such as antigen expression and presentation, the varying HLA restrictions between different individuals, and the potential for viral escape by sequence variation or surface molecule alteration on infected cells. A strategy to circumvent these limitations is the construction of a chimeric T cell receptor containing human CD4 or HIV-1-specific Ig sequences linked to the signaling domain of the T cell receptor ζ chain (universal T cell receptor). CD8+CTLs transduced with this universal receptor can then bind and lyse infected cells that express surface HIV-1 gp120. We evaluated the ability of universal-receptor-bearing CD8+cells from a seronegative donor to lyse acutely infected cells and inhibit HIV-1 replicationin vitro. The kinetics of lysis and efficiency of inhibition were comparable to that of naturally occurring HIV-1-specific CTL clones isolated from infected individuals. Further study will be required to determine the utility of these cells as a therapeutic strategyin vivo.
DHR
3, an ecdysone-inducible early-late gene encoding a
Drosophila
nuclear receptor, is required for embryogenesis
Authors: Carney Ginger E. , Wade Andrew A. , Sapra Rajat , Goldstein Elliott S. , Bender Michael
Published: July 2002

Response to the steroid hormone ecdysone in
Drosophila
is controlled by genetic regulatory hierarchies that include eight members of the nuclear receptor protein family. The
DHR3
gene, located within the 46F early-late ecdysone-inducible chromosome puff, encodes an orphan nuclear receptor that recently has been shown to exert both positive and negative regulatory effects in the ecdysone-induced genetic hierarchies at metamorphosis. We used a reverse genetics approach to identify 11
DHR3
mutants from a pool of lethal mutations in the 46F region on the second chromosome. Two
DHR3
mutations result in amino acid substitutions within the conserved DNA binding domain. Analysis of
DHR3
mutants reveals that
DHR3
function is required to complete embryogenesis. All
DHR3
alleles examined result in nervous system defects in the embryo.
Aneuploidy correlated 100% with chemical transformation of Chinese hamster cells
Authors: Li Ruhong , Yerganian George , Duesberg Peter , Kraemer Alwin , Willer Andreas , Rausch Charlotte , Hehlmann Ruediger
Published: July 2002

Aneuploidy or chromosome imbalance is the most massive genetic abnormality of cancer cells. It used to be considered the cause of cancer when it was discovered more than 100 years ago. Since the discovery of the gene, the aneuploidy hypothesis has lost ground to the hypothesis that mutation of cellular genes causes cancer. According to this hypothesis, cancers are diploid and aneuploidy is secondary or nonessential. Here we reexamine the aneuploidy hypothesis in view of the fact that nearly all solid cancers are aneuploid, that many carcinogens are nongenotoxic, and that mutated genes from cancer cells do not transform diploid human or animal cells. By regrouping the gene pool—as in speciation—aneuploidy inevitably will alter many genetic programs. This genetic revolution can explain the numerous unique properties of cancer cells, such as invasiveness, dedifferentiation, distinct morphology, and specific surface antigens, much better than gene mutation, which is limited by the conservation of the existing chromosome structure. To determine whether aneuploidy is a cause or a consequence of transformation, we have analyzed the chromosomes of Chinese hamster embryo (CHE) cells transformedin vitro. This system allows (i) detection of transformation within 2 months and thus about 5 months sooner than carcinogenesis and (ii) the generation of many more transformants per cost than carcinogenesis. To minimize mutation of cellular genes, we have used nongenotoxic carcinogens. It was found that 44 out of 44 colonies of CHE cells transformed by benz[a]pyrene, methylcholanthrene, dimethylbenzanthracene, and colcemid, or spontaneously were between 50 and 100% aneuploid. Thus, aneuploidy originated with transformation. Two of two chemically transformed colonies tested were tumorigenic 2 months after inoculation into hamsters. The cells of transformed colonies were heterogeneous in chromosome number, consistent with the hypothesis that aneuploidy can perpetually destabilize the chromosome number because it unbalances the elements of the mitotic apparatus. Considering that all 44 transformed colonies analyzed were aneuploid, and the early association between aneuploidy, transformation, and tumorigenicity, we conclude that aneuploidy is the cause rather than a consequence of transformation.
Expression genetics in cancer: Shifting the focus from DNA to RNA
Authors: Sager Ruth
Published: July 2002

Expression genetics is a conceptually different approach to the identification of cancer-related genes than the search for mutations at the genome level. While mutations lie at the heart of cancer, at least in its early stages, what is recognized here are phenotypic changes usually many steps removed from the initiating mutation. Classically cancer geneticists have concentrated on genomic changes and have ignored the productive potential of examining downstream events based on screening for differential gene expression between tumor cells and well matched normal counterparts. Genes involved in cancer affect the normal functions of many cellular processes: not only proliferation but cell–cell and cell–matrix interactions, DNA repair, invasion and motility, angiogenesis, senescence, apoptosis, and others. Yet very few cancer-related genes affecting these processes have been identified in human cancers by classical methods to find mutated genes despite enormous efforts. I report here our success in readily isolating more than 100 candidate tumor suppressor genes from human tissue, estimated to represent roughly 20% of the total genes recoverable by this approach. Half of the genes are unknown and the other half include representatives of most known cancer processes. Because their expression is lost during cancer progression, they may be useful tumor markers for diagnosis and prognosis. Because these genes are not mutated, they provide opportunities for pharmacological intervention by inducing their reexpression.
Blockage of muscle and neuronal nicotinic acetylcholine receptors by fluoxetine (Prozac)
Authors: García-Colunga J. , Awad J. N. , Miledi R.
Published: July 2002

Fluoxetine (Prozac), a widely used antidepressant, is said to exert its medicinal effects almost exclusively by blocking the serotonin uptake systems. The present study shows that both muscle and neuronal nicotinic acetylcholine receptors are blocked, in a noncompetitive and voltage-dependent way, by fluoxetine, which also increases the rate of desensitization of the nicotinic receptors. Because these receptors are very widely distributed in the both central and peripheral nervous systems, the blocking action of fluoxetine on nicotinic receptors may play an important role in its antidepressant and other therapeutical effects. Our findings will help to understand the mode of action of fluoxetine, and they may also help to develop more specific medicinal drugs.
Nucleosome-mediated synergism between transcription factors on the mouse mammary tumor virus promoter
Authors: Chávez Sebastián , Beato Miguel
Published: July 2002

In unstimulated mammalian cells and in
Saccharomyces cerevisiae
, the mouse mammary tumor virus (MMTV) promoter is silent and organized into positioned nucleosomes, one of which encompasses the binding sites for glucocorticoid receptor (GR) and nuclear factor I (NFI). Glucocorticoid induction
in vivo
involves a functional synergism between GR and NFI and simultaneous occupancy of the promoter sites for both proteins that cannot be reproduced on naked DNA. The role of chromatin in the process of induction was investigated by manipulating the nucleosome density in yeast strains carrying a regulated histone H4 gene. Following depletion of nucleosomes, independent transactivation by NFI or by GR, as well as binding of the individual proteins to the MMTV promoter, were enhanced, in agreement with a repressive function of nucleosomes. In contrast, NFI-dependent hormone induction of the promoter and the simultaneous binding of receptor and NFI were compromised by nucleosome depletion. This effect could be partly mediated by a cryptic binding site for the receptor that is functional only in the nucleosomal context. Thus, positioned nucleosomes do not only account for constitutive repression of the MMTV promoter, but also participate in induction by mediating cooperative binding and functional synergism between GR and NFI.
Inhibition of
DLX-7
homeobox gene causes decreased expression of
GATA-1
and c-
myc
genes and apoptosis
Authors: Shimamoto Takashi , Nakamura Shuji , Bollekens Jacques , Ruddle Frank H. , Takeshita Kenichi
Published: July 2002

The
DLX
gene family is a family of divergent homeobox genes which are related to the
Drosophila
distal-less (
Dll
) gene and has been reported to be expressed primarily in the forebrain and craniofacial structures. We have previously identified a new member of this family,
DLX-7
. We now report that this gene is expressed in normal hematopoietic cells and leukemia cell lines with erythroid characteristics. We used an antisense oligonucleotide targeted against the translation start site of
DLX-7
mRNA to inhibit its expression in a human erythroleukemia cell line K562, which expresses
DLX-7
at a high level. The antisense oligonucleotide efficiently reduced the
DLX-7
mRNA, while control oligonucleotides, including a mutant oligonucleotide identical to the antisense sequence except for four nucleotide mismatches, had no effect on
DLX-7
mRNA level. Inhibition of
DLX-7
expression decreased the plating efficiency by ≈70% compared with control. The antisense treatment caused apoptosis, as shown by the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-digoxigenin nick end labeling (TUNEL) method. Down-regulation of
DLX-7
expression by antisense treatment was associated with a reduction in
GATA-1
and c-
myc
mRNA levels. Thus, we conclude that
DLX-7
is expressed in hematopoietic cells and that the inhibition of its expression results in the decreased levels of
GATA-1
and c-
myc
genes, with an accompanying induction of apoptosis.
The
nude
gene encodes a sequence-specific DNA binding protein with homologs in organisms that lack an anticipatory immune system
Authors: Schlake Thomas , Schorpp Michael , Nehls Michael , Boehm Thomas
Published: July 2002

In the mouse, the product of the
nude
locus, Whn, is required for the keratinization of the hair shaft and the differentiation of epithelial progenitor cells in the thymus. A bacterially expressed peptide representing the presumptive DNA binding domain of the mouse
whn
gene
in vitro
specifically binds to a 11-bp consensus sequence containing the invariant tetranucleotide 5′-ACGC. In transient transfection assays, such binding sites stimulated reporter gene expression about 30- to 40-fold, when positioned upstream of a minimal promotor. Whn homologs from humans, bony fish (
Danio rerio
), cartilaginous fish (
Scyliorhinus caniculus
), agnathans (
Lampetra planeri
), and cephalochordates (
Branchiostoma lanceolatum
) share at least 80% of amino acids in the DNA binding domain. In agreement with this remarkable structural conservation, the DNA binding domains from zebrafish, which possesses a thymus but no hair, and amphioxus, which possesses neither thymus nor hair, recognize the same target sequence as the mouse DNA binding domain
in vitro
and
in vivo
. The genomes of vertebrates and cephalochordates contain only a single
whn
-like gene, suggesting that the primordial
whn
gene was not subject to gene-duplication events. Although the role of
whn
in cephalochordates and agnathans is unknown, its requirement in the development of the thymus gland and the differentiation of skin appendages in the mouse suggests that changes in the transcriptional control regions of
whn
genes accompanied their functional reassignments during evolution.
Reduced RNA polymerase II transcription in intact and permeabilized Cockayne syndrome group B cells
Authors: Balajee Adayabalam S. , May Alfred , Dianov Grigory L. , Friedberg Errol C. , Bohr Vilhelm A.
Published: July 2002

Cockayne syndrome (CS) is characterized by increased photosensitivity, growth retardation, and neurological and skeletal abnormalities. The recovery of RNA synthesis is abnormally delayed in CS cells after exposure to UV radiation. Gene-specific repair studies have shown a defect in the transcription-coupled repair (TCR) of active genes in CS cells from genetic complementation groups A and B (CS-A and CS-B). We have analyzed transcription
in vivo
in intact and permeabilized CS-B cells. Uridine pulse labeling in intact CS-B fibroblasts and lymphoblasts shows a reduction of ≈50% compared with various normal cells and with cells from a patient with xeroderma pigmentosum (XP) group A. In permeabilized CS-B cells transcription in chromatin isolated under physiological conditions is reduced to about 50% of that in normal chromatin and there is a marked reduction in fluorescence intensity in transcription sites in interphase nuclei. Transcription in CS-B cells is sensitive to α-amanitin, suggesting that it is RNA polymerase II-dependent. The reduced transcription in CS-B cells is complemented in chromatin by the addition of normal cell extract, and in intact cells by transfection with the
CSB
gene. CS-B may be a primary transcription deficiency.