bluuebunny/crossref_metadata_2025 huggingface dataset:
Isolation and characterization of a tobacco mosaic virus-inducible
myb
oncogene homolog from tobacco
Authors: Yang Yinong , Klessig Daniel F.
Published: July 2002

Salicylic acid (SA) plays an important role in signaling the
activation of plant defense responses against pathogen attack including
induction of pathogenesis-related (PR) proteins. To gain further
insight into the SA-mediated signal transduction pathway, we have
isolated and characterized a tobacco mosaic virus (TMV)-inducible
myb
oncogene homolog (
myb1
) from tobacco.
The
myb1
gene was induced upon TMV infection during both
the hypersensitive response and development of systemic acquired
resistance in the resistant tobacco cultivar following the rise of
endogenous SA, but was not activated in the susceptible
cultivar that fails to accumulate SA. The
myb1
gene was
also induced by incompatible bacterial pathogen
Pseudomonas
syringae
pv. syringae during the hypersensitive response.
Exogenous SA treatment rapidly (within 15 min) activated the expression
of
myb1
in both resistant and susceptible tobacco
cultivars with the subsequent induction of PR genes occurring several
hours later. Biologically active analogs of SA and
2,6-dichloroisonicotinic acid (a synthetic functional analog of SA),
which induce PR genes and enhanced resistance, also activated the
myb1
gene. In contrast, biologically inactive analogs
were poor inducers of
myb1
gene expression. Furthermore,
the recombinant Myb1 protein was shown to specifically bind to a
Myb-binding consensus sequence found in the promoter of the
PR-1a
gene. Taken together, these results suggest that
the tobacco
myb1
gene encodes a signaling component
downstream of SA that may participate in transcriptional activation of
PR genes and plant disease resistance.
The Malthusian parameter of ascents: What prevents the exponential increase of one’s ancestors?
Authors: Ohno Susumu
Published: July 2002

The reason that the indefinite exponential increase in the number
of one’s ancestors does not take place is found in the law of sibling
interference, which can be expressed by the following simple
equation:

\documentclass[12pt]{minimal}
\usepackage{amsmath}
\usepackage{wasysym}
\usepackage{amsfonts}
\usepackage{amssymb}
\usepackage{amsbsy}
\usepackage{mathrsfs}
\setlength{\oddsidemargin}{-69pt}
\begin{document}
\begin{equation*}\begin{matrix}{\mathit{N}}_{{\mathit{n}}} \enskip & \\ {\mathit{{\blacksquare}}} \enskip & \\ {\mathit{ASZ}} \enskip & \end{matrix}
{\mathrm{\hspace{.167em}{\times}\hspace{.167em}2\hspace{.167em}=\hspace{.167em}}}{\mathit{N_{n+1},}}\end{equation*}\end{document}

where
N

n

is the number of ancestors in the
n
th generation,
ASZ
is the average sibling size
of these ancestors, and
N

n

+1
is the
number of ancestors in the next older generation (
n
+ 1).
Accordingly, the exponential increase in the number of one’s ancestors
is an initial anomaly that occurs while
ASZ
remains at 1.
Once
ASZ
begins to exceed 1, the rate of increase in the
number of ancestors is progressively curtailed, falling further and
further behind the exponential increase rate. Eventually,
ASZ
reaches 2, and at that point, the number of ancestors
stops increasing for two generations. These two generations, named AN
SA and AN SA + 1, are the most critical in the ancestry, for one’s
ancestors at that point come to represent all the progeny-produced
adults of the entire ancestral population. Thereafter, the fate of
one’s ancestors becomes the fate of the entire population. If the
population to which one belongs is a successful, slowly expanding one,
the number of ancestors would slowly decline as you move toward the
remote past. This is because
ABZ
would exceed 2. Only when
ABZ
is less than 2 would the number of ancestors increase
beyond the AN SA and AN SA + 1 generations. Since the above is an
indication of a failing population on the way to extinction, there had
to be the previous AN SA involving a far greater number of individuals
for such a population. Simulations indicated that for a member of a
continuously successful population, the AN SA ancestors might have
numbered as many as 5.2 million, the AN SA generation being the 28th
generation in the past. However, because of the law of increasingly
irrelevant remote ancestors, only a very small fraction of the AN SA
ancestors would have left genetic traces in the genome of each
descendant of today.
Genes encoding the same three subunits of respiratory complex II are present in the mitochondrial DNA of two phylogenetically distant eukaryotes.
Authors: Burger G , Lang B F , Reith M , Gray M W
Published: July 2002

Although mitochondrial DNA is known to encode a limited number (<20) of the polypeptide components of respiratory complexes I, III, IV, and V, genes for components of complex II [succinate dehydrogenase (ubiquinone); succinate:ubiquinone oxidoreductase, EC 1.3.5.1] are conspicuously lacking in mitochondrial genomes so far characterized. Here we show that the same three subunits of complex II are encoded in the mitochondrial DNA of two phylogenetically distant eukaryotes, Porphyra purpurea (a photosynthetic red alga) and Reclinomonas americana (a heterotrophic zooflagellate). These complex II genes, sdh2, sdh3, and sdh4, are homologs, respectively, of Escherichia coli sdhB, sdhC, and sdhD. In E. coli, sdhB encodes the iron-sulfur subunit of succinate dehydrogenase (SDH), whereas sdhC and sdhD specify, respectively, apocytochrome b558 and a hydrophobic 13-kDa polypeptide, which together anchor SDH to the inner mitochondrial membrane. Amino acid sequence similarities indicate that sdh2, sdh3, and sdh4 were originally encoded in the protomitochondrial genome and have subsequently been transferred to the nuclear genome in most eukaryotes. The data presented here are consistent with the view that mitochondria constitute a monophyletic lineage.
Design of polydactyl zinc-finger proteins for unique addressing within complex genomes
Authors: Liu Qiang , Segal David J. , Ghiara Jayant B. , Barbas Carlos F.
Published: July 2002

Zinc-finger proteins of the Cys
2
-His
2
type represent a class of malleable DNA-binding proteins that may be selected to bind diverse sequences. Typically, zinc-finger proteins containing three zinc-finger domains, like the murine transcription factor Zif268 and the human transcription factor Sp1, bind nine contiguous base pairs. To create a class of proteins that would be generally applicable to target unique sites within complex genomes, we have utilized structure-based modeling to design a polypeptide linker that fuses two three-finger proteins. Two six-fingered proteins were created and demonstrated to bind 18 contiguous bp of DNA in a sequence-specific fashion. Expression of these proteins as fusions to activation or repression domains allows transcription to be specifically up- or down-modulated within human cells. Polydactyl zinc-finger proteins should be broadly applicable as genome-specific transcriptional switches in gene therapy strategies and the development of novel transgenic plants and animals.
Conditional activation defect of a human G
sα
mutant
Authors: Iiri Taroh , Farfel Zvi , Bourne Henry R.
Published: July 2002

Hormonal signals activate trimeric G proteins by promoting exchange of GTP for GDP bound to the G protein’s α subunit (Gα). Here we describe a point mutation that impairs this activation mechanism in the α subunit of G
s
, producing an inherited disorder of hormone responsiveness. Biochemical analysis reveals an activation defect that is paradoxically intensified by hormonal and other stimuli. By substituting histidine for a conserved arginine residue, the mutation removes an internal salt bridge (to a conserved glutamate) that normally acts as an intramolecular hasp to maintain tight binding of the γ-phosphate of GTP. In its basal, unperturbed state, the mutant α
s
binds guanosine 5′-[γ-thio]triphosphate (GTP[γS]), a GTP analog, slightly less tightly than does normal α
s
, but (in the GTP[γS]-bound form) can stimulate adenylyl cyclase. The activation defect becomes prominent only under conditions that destabilize binding of guanine nucleotide (receptor stimulation) or impair the ability of α
s
to bind the γ-phosphate of GTP (cholera toxin, AlF
4
−
ion). Although GDP release is usually the rate-limiting step in nucleotide exchange, the biochemical phenotype of this mutant α
s
indicates that efficient G protein activation by receptors and other stimuli depends on the ability of Gα to clasp tightly the GTP molecule that enters the binding site.
Inducible gene expression and protein translocation using nontoxic ligands identified by a mammalian three-hybrid screen
Authors: Liberles Stephen D. , Diver Steven T. , Austin David J. , Schreiber Stuart L.
Published: July 2002

The natural product rapamycin has been used to provide temporal and quantitative control of gene expression in animals through its ability to interact with two proteins simultaneously. A shortcoming of this approach is that rapamycin is an inhibitor of cell proliferation, the result of binding to FKBP12–rapamycin-associated protein (FRAP). To overcome this limitation, nontoxic derivatives of rapamycin bearing bulky substituents at its C16-position were synthesized, each in a single step. The isosteric isopropoxy and methallyl substituents with the nonnatural C16-configuration abolish both binding to FRAP and inhibition of T cell proliferation. Binding proteins for these derivatives were identified from libraries of cDNAs encoding mutants of the FKBP12–rapamycin-binding (FRB) domain of FRAP by using a mammalian three-hybrid transcription assay. Targeting of the mutations was guided by the structure of the FKBP12-rapamycin–FRB ternary complex. Three compensatory mutations in the FRB domain, all along one face of an α-helix in a rapamycin-binding pocket, were identified that together restore binding of the rapamycin derivatives. Using this mutant FRB domain, one of the nontoxic rapamycin derivatives induced targeted gene expression in Jurkat T cells with an EC
50
below 10 nM. Another derivative was used to recruit a cytosolic protein to the plasma membrane, mimicking a process involved in many signaling pathways.
A p53-independent damage-sensing mechanism that functions as a checkpoint at the G
1
/S transition in Chinese hamster ovary cells
Authors: Lee Hoyun , Larner James M. , Hamlin Joyce L.
Published: July 2002

In response to a moderate dose of radiation, asynchronous mammalian cell populations rapidly and transiently down-regulate the rate of DNA synthesis to ≈50% of preirradiation values. We show here that only half of the reduction in overall replication rate can be accounted for by direct inhibition of initiation at origins in S-phase cells. The other half results from the operation of a newly defined cell cycle checkpoint that functions at the G
1
/S transition. This checkpoint senses damage incurred at any time during the last 2 hr of G
1
and effectively prevents entry into the S period. The G
1
/S and S-phase checkpoints are both p53-independent and, unlike the p53-mediated G
1
checkpoint, respond rapidly to radiation, suggesting that they may represent major damage-sensing mechanisms connecting the replication machinery with DNA repair pathways.
Terpenoid biosynthesis from 1-deoxy-
d
-xylulose in higher plants by intramolecular skeletal rearrangement
Authors: Arigoni Duilio , Sagner Silvia , Latzel Christoph , Eisenreich Wolfgang , Bacher Adelbert , Zenk Meinhart H.
Published: July 2002

The incorporation of [1-
13
C]- and [2,3,4,5-
13
C
4
]1-deoxy-
d
-xylulose into β-carotene, lutein, phytol, and sitosterol in a cell culture of
Catharanthus roseus
was analyzed by NMR spectroscopy. The labeling patterns of the isoprene precursors, isopentenyl pyrophosphate and dimethylallyl pyrophosphate, were obtained from the terpenes by a retrobiosynthetic approach.
13
C Enrichment and
13
C
13
C coupling patterns showed conclusively that 1-deoxy-
d
-xylulose and not mevalonate is the predominant isoprenoid precursor of phytol, β-carotene, and lutein. Label from 1-deoxyxylulose was also diverted to phytosterols to a minor extent (6% relative to carotene and phytol formation). The data demonstrate that the formation of isopentenyl pyrophosphate from pentulose occurs strictly by an intramolecular rearrangement process.
Selective induction of p53 and chemosensitivity in RB-deficient cells by E1A mutants unable to bind the RB-related proteins
Authors: Samuelson Andrew V. , Lowe Scott W.
Published: July 2002

The adenovirus E1A oncoprotein renders primary cells sensitive to the induction of apoptosis by diverse stimuli, including many anticancer agents. E1A-expressing cells accumulate p53 protein, and p53 potentiates drug-induced apoptosis. To determine how E1A promotes chemosensitivity, a series of E1A mutants were introduced into primary human and mouse fibroblasts using high-titer recombinant retroviruses, allowing analysis of E1A in genetically normal cells outside the context of adenovirus infection. Mutations that disrupted apoptosis and chemosensitivity separated into two complementation groups, which correlated precisely with the ability of E1A to associate with either the p300/CBP or retinoblastoma protein families. Furthermore, E1A mutants incapable of binding RB, p107, and p130 conferred chemosensitivity to fibroblasts derived from
RB
-deficient mice, but not fibroblasts from mice lacking p107 or p130. Hence, inactivation of RB, but not p107 or p130, is required for chemosensitivity induced by E1A. Finally, the same E1A functions that promote drug-induced apoptosis also induce p53. Together, these data demonstrate that p53 accumulation and chemosensitivity are linked to E1A’s oncogenic potential, and identify a strategy to selectively induce apoptosis in RB-deficient tumor cells.
Expression of hepatocyte nuclear factor 6 in rat liver is sex-dependent and regulated by growth hormone
Authors: Lahuna Olivier , Fernandez Leandro , Karlsson Hanna , Maiter Dominique , Lemaigre Frederic P. , Rousseau Guy G. , Gustafsson Jan-Åke , Mode Agneta
Published: July 2002

Growth hormone (GH) binding to its receptor modulates gene transcription by influencing the amount or activity of transcription factors. In the rat, GH exerts sexually dimorphic effects on liver gene transcription through its pattern of secretion which is intermittent in males and continuous in females. The expression of the
CYP2C12
gene coding for the female-specific cytochrome P450 2C12 protein is dependent on the continuous exposure to GH. To identify the transcription factor(s) that mediate(s) this sex-dependent GH effect, we studied the interactions of the
CYP2C12
promoter with liver nuclear proteins obtained from male and female rats and from hypophysectomized animals treated or not by continuous GH infusion. GH treatment induced the binding of a protein that we identified as hepatocyte nuclear factor (HNF) 6, the prototype of a novel class of homeodomain transcription factors. HNF-6 competed with HNF-3 for binding to the same site in the
CYP2C12
promoter. This HNF-6/HNF-3 binding site conveyed both HNF-6- and HNF-3-stimulated transcription of a reporter gene construct in transient cotransfection experiments. Electrophoretic mobility shift assays showed more HNF-6 DNA-binding activity in female than in male liver nuclear extracts. Liver HNF-6 mRNA was barely detectable in the hypophysectomized rats and was restored to normal levels by GH treatment. This work provides an example of a homeodomain-containing transcription factor that is GH-regulated and also reports on the hormonal regulation of HNF-6.
Generation of NK1.1+T cell antigen receptor α/β+thymocytes associated with intact thymic structure
Authors: Nakagawa Ken-ichi , Iwabuchi Kazuya , Ogasawara Kazumasa , Ato Manabu , Kajiwara Masaharu , Nishihori Hiroki , Iwabuchi Chikako , Ishikura Hiroshi , Good Robert A. , Onoé Kazunori
Published: July 2002

The development of T cells within the thymus is largely dependent on intact cortical and medullary epithelial cells. However, it has been reported that positive selection of natural killer antigen 1.1+(NK1.1+) T cell antigen receptor (TCR)-α/β+thymocytes recently identified among CD4+8−and CD4−8−subpopulations is attributable to major histocompatibility complex class Ib ligands expressed on bone marrow (BM)-derived components in the thymus. In the present study, we investigated generation of NK1.1+TCR-α/β+cells in the thymus of thealy/alymouse which lacks lymph nodes and Peyer’s patches and shows abnormalities of thymic and splenic structure. We found that the proportion of the NK1.1+TCR-α/β+thymocytes was extremely low in these mice as compared withaly/+ and normal C57BL/6 mice. Thymic reconstitution by BM cells fromaly/+ mice that possess a normal population of NK1.1+TCR-α/β+cell population did not restore the NK1.1+TCR-α/β+cell population in the thymus of lethally irradiatedaly/alymouse. When deoxyguanosine-treated fetal thymi from (B6 × B10.G)F1mice were transplanted toaly/alymice that had been thymectomized and reconstituted with BM cells ofaly/alymice, normal proportions of the NK1.1+TCR-α/β+thymocytes were present in the thymus grafts. These findings demonstrate that the development of NK1.1+TCR-α/β+thymocytes is accomplished under the influence not only of BM-derived components, but also of irradiation-resistant or deoxyguanosine-resistant components and an intact microenvironment of the thymus.
Cloning and characterization of the extreme 5′-terminal sequences of the RNA genomes of GB virus C/hepatitis G virus
Authors: Hsieh Sen-Yung , Yang Pei-Ying , Chen Hsiao-Chin , Liaw Yun-Fan
Published: July 2002

The extreme 5′-terminal sequences of the GB virus C/hepatitis G virus (GBV-C/HGV), containing elements essential for regulation of viral gene expression and replication, have not been determined. By using a RNA-ligase-mediated RACE (rapid amplification of the cDNA ends) procedure, we have cloned the extreme 5′-terminal sequences of the viral genome from the serum of three Taiwanese patients. Sequence analysis of the 5′ noncoding region in alignment with one West African and two American isolates showed that (
i
) a consensus 5′-end sequence was cloned; (
ii
) about 97% of sequences were homologous among the three Taiwan isolates and also between the two American isolates, whereas about 90% of sequences were homologous among the isolates from the three different geographic areas; (
iii
) the sequence heterogeneity related to geographic separation is confined mainly to three domains; and (
iv
) a potential hairpin structure, resembling the hairpin structure found in the 5′ end of hepatitis C virus genome, was detected in the 5′ end of the noncoding region. Our data support the hypotheses that (
i
) the extreme 5′ end of the hepatitis GBV-C/HGV viral genome has been cloned, (
ii
) there are different genotypes correlated with geographic separation, and (
iii
) the viral translation and replication mechanisms may be similar to that of hepatitis C virus and pestiviruses. Our data have not only shed light on the viral replication mechanism but also offer information for selection of optimal primer sequences for the detection and genotyping of the hepatitis GBV-C/HGV virus by PCR assays.
Two distinct populations of synaptic-like vesicles from rat brain
Authors: Thoidis Galini , Chen Peng , Pushkin Alexander V. , Vallega Gino , Leeman Susan E. , Fine Richard E. , Kandror Konstantin V.
Published: July 2002

In nonneuronal cells, several plasma membrane proteins such as exofacial enzymes, receptors, and ion channels recycle between their intracellular compartment(s) and the cell surface via an endosomal pathway. In neurons, however, this pathway has not been extensively characterized. In particular, it remains unclear whether or not it is related to the recycling of small synaptic vesicles, the major pathway of membrane traffic in nerve terminals. To approach this problem, we purified and studied a vesicular fraction from rat brain synaptosomes. Two distinct populations of vesicles with different buoyant densities and sedimentation coefficients were detected in this fraction by sucrose gradient centrifugation and Western blot analysis of the individual proteins. Both populations contain proteins that are markers of synaptic vesicles, namely, SV2, synaptotagmin, synaptophysin, secretory carrier membrane proteins (SCAMPs), synaptobrevin, and rab3a. A striking difference between the two populations is the presence of arginine aminopeptidase activity (a previously suggested marker for the regulated endosomal recycling pathway) exclusively in the lighter less-dense vesicles. The same two vesicular populations were also detected in the preparation of clathrin-coated vesicles isolated from whole rat brain or purified synaptosomes after removal of their clathrin coats by incubation at pH 8.5. We conclude, therefore, that both types of vesicles recycle in synaptosomes via a clathrin-mediated pathway. These data present experimental evidence for biochemical heterogeneity of synaptic-like vesicles in rat brain.
Evidence of high expression of peptidylglycine α-amidating monooxygenase in the rat uterus: Estrogen regulation
Authors: Meskini Rajaâ El , Delfino Christine , Boudouresque Françoise , Oliver Charles , Martin Pierre-Marie , Ouafik L’Houcine
Published: July 2002

In the present study, high levels of peptidylglycine α-amidating monooxygenase (PAM), which catalyzes the two-step formation of bioactive α-amidated peptides from their glycine-extended precursors, have been found in the uterus. Expression of PAM was evaluated in the uterus of intact cycling adult female rats and after experimental manipulation of the estrogen status of the rats. During the estrous cycle, PAM mRNA levels exhibited striking changes inversely related to the physiological variations of plasma estrogen levels. The levels of PAM transcripts changed markedly during the estrous cycle, reaching the highest levels at metestrus. There was a 15-fold increase in the abundance of PAM mRNA between metestrus and proestrus. Chronic treatment of ovariectomized rats with 17β-estradiol decreased PAM mRNA levels to values comparable with those found in intact rats at proestrus. Progesterone was without effect on PAM mRNA levels, indicating that the effect was specific for estradiol.
In situ
hybridization studies were conducted to determine the tissue disposition and cell types expressing PAM. High levels of PAM mRNA were localized in the endometrium at the level of luminal and glandular cells. A weak signal was observed in stromal cells, and the myometrium cells were negative. 17β-Estradiol treatment induced an overall decrease of the hybridization signal, as compared with ovariectomized rats. These results demonstrate the presence of high levels of PAM in the uterus and indicate that estrogens are involved in regulating the expression of the enzyme in this tissue. However, the present study provides no information regarding whether this regulation takes place at the level of transcription or influences mRNA stability.
Neurabin is a synaptic protein linking p70 S6 kinase and the neuronal cytoskeleton
Authors: Burnett Patrick E. , Blackshaw Seth , Lai Michael M. , Qureshi Irfan A. , Burnett Aaron F. , Sabatini David M. , Snyder Solomon H.
Published: July 2002

p70 S6 kinase (p70
S6k
) is a mitogen-activated protein kinase that plays a central role in the control of mRNA translation. It physiologically phosphorylates the S6 protein of the 40s ribosomal subunit in response to mitogenic stimuli and is a downstream component of the rapamycin-sensitive pathway, which includes the 12-kDa FK506 binding protein and includes rapamycin and the 12-kDa FK506 binding protein target 1. Here, we report the identification of neurabin (neural tissue-specific F-actin binding protein), a neuronally enriched protein of 1,095 amino acids that contains a PDZ domain and binds p70
S6k
. We demonstrate the neurabin-p70
S6k
interaction by yeast two-hybrid analysis and biochemical techniques. p70
S6k
and neurabin coimmunoprecipitate from transfected HEK293 cells. Site-directed mutagenesis of neurabin implicates its PDZ domain in the interaction with p70
S6k
, and deletion of the carboxyl-terminal five amino acids of p70
S6k
abrogates the interaction. Cotransfection of neurabin in HEK293 cells activates p70
S6k
kinase activity. The mRNA of neurabin and p70
S6k
show striking colocalization in brain sections by
in situ
hybridization. Subcellular fractionation of rat brain demonstrates that neurabin and p70
S6k
both localize to the soluble fraction of synaptosomes. By way of its PDZ domain, the neuronal-specific neurabin may target p70
S6k
to nerve terminals.
Consistent, persistent expression from modified retroviral vectors in murine hematopoietic stem cells
Authors: Robbins Paul B. , Skelton Dianne C. , Yu Xiao-Jin , Halene Stephanie , Leonard Earl H. , Kohn Donald B.
Published: July 2002

Retroviral vectors based on the Moloney murine leukemia virus (MoMuLV) have shown inconsistent levels and duration of expression as well as a propensity for the acquisition of
de novo
methylation
in vivo
. MoMuLV-based vectors are known to contain sequences that are capable of suppressing or preventing expression from the long terminal repeat. Previously, we constructed a series of modified retroviral vectors and showed that they function significantly better than MoMuLV-based vectors
in vitro
. To test the efficacy of the modified vectors in hematopoietic stem cells
in vivo,
we examined gene expression and proviral methylation in differentiated hematopoietic colonies formed in the spleens of mice after serial transplantation with transduced bone marrow (2°CFU-S). We found a significant increase in the frequency of expression with our modified vectors (>90% expression in vector DNA containing 2°CFU-S) over the frequency observed with the standard MoMuLV-based vector (28% expression in vector containing 2°CFU-S). Expression from the modified vectors was highly consistent, with expression in >50% of the vector-containing 2°CFU-S from all 20 transplant recipients analyzed, whereas expression from the standard MoMuLV-based vector was inconsistent, with expression in 0–10% of the vector containing 2°CFU-S from 8 recipients and expression in >50% of the vector-containing 2°CFU-S from 4 other recipients. In addition, we established that the modified vectors had a lower level of DNA methylation than the control vector. These findings represent significant advances in the development and evaluation of effective retroviral vectors for application
in vivo
.
Rapid noninvasive detection of experimental atherosclerotic lesions with novel
99m
Tc-labeled diadenosine tetraphosphates
Authors: Elmaleh David R. , Narula Jagat , Babich John W. , Petrov Artiom , Fischman Alan J. , Khaw Ban-An , Rapaport Eliezer , Zamecnik Paul C.
Published: July 2002

The development of a noninvasive imaging procedure for identifying atherosclerotic lesions is extremely important for the clinical management of patients with coronary artery and peripheral vascular disease. Although numerous radiopharmaceuticals have been proposed for this purpose, none has demonstrated the diagnostic accuracy required to replace invasive angiography. In this report, we used the radiolabeled purine analog,
99m
Tc diadenosine tetraphosphate (Ap
4
A; AppppA,
P
1
,
P
4
-di(adenosine-5′)-tetraphosphate) and its analogue
99m
Tc AppCHClppA for imaging experimental atherosclerotic lesions in New Zealand White rabbits. Serial gamma camera images were obtained after intravenous injection of the radiolabeled dinucleotides. After acquiring the final images, the animals were sacrificed,
ex vivo
images of the aortas were recorded, and biodistribution was measured.
99m
Tc-Ap
4
A and
99m
Tc AppCHClppA accumulated rapidly in atherosclerotic abdominal aorta, and lesions were clearly visible within 30 min after injection in all animals that were studied. Both radiopharmaceuticals were retained in the lesions for 3 hr, and the peak lesion to normal vessel ratio was 7.4 to 1. Neither of the purine analogs showed significant accumulation in the abdominal aorta of normal (control) rabbits. The excised aortas showed lesion patterns that were highly correlated with the
in vivo
and
ex vivo
imaging results. The present study demonstrates that purine receptors are up-regulated in experimental atherosclerotic lesions and
99m
Tc-labeled purine analogs have potential for rapid noninvasive detection of plaque formation.
Crystal structure of
Taq
DNA polymerase in complex with an inhibitory Fab: The Fab is directed against an intermediate in the helix-coil dynamics of the enzyme
Authors: Murali R. , Sharkey D. J. , Daiss J. L. , Murthy H. M. Krishna
Published: July 2002

We report the crystal structure of
Thermus aquaticus
DNA polymerase I in complex with an inhibitory Fab, TP7, directed against the native enzyme. Some of the residues present in a helical conformation in the native enzyme have adopted a γ turn conformation in the complex. Taken together, structural information that describes alteration of helical structure and solution studies that demonstrate the ability of TP7 to inhibit 100% of the polymerase activity of the enzyme suggest that the change in conformation is probably caused by trapping of an intermediate in the helix-coil dynamics of this helix by the Fab. Antibodies directed against modified helices in proteins have long been anticipated. The present structure provides direct crystallographic evidence. The Fab binds within the DNA binding cleft of the polymerase domain, interacting with several residues that are used by the enzyme in binding the primer:template complex. This result unequivocally corroborates inferences drawn from binding experiments and modeling calculations that the inhibitory activity of this Fab is directly attributable to its interference with DNA binding by the polymerase domain of the enzyme. The combination of interactions made by the Fab residues in both the polymerase and the vestigial editing nuclease domain of the enzyme reveal the structural basis of its preference for binding to DNA polymerases of the
Thermus
species. The orientation of the structure-specific nuclease domain with respect to the polymerase domain is significantly different from that seen in other structures of this polymerase. This reorientation does not appear to be antibody-induced and implies remarkably high relative mobility between these two domains.
Long-term but not short-term plasticity at mossy fiber synapses is impaired in neural cell adhesion molecule-deficient mice
Authors: Cremer Harold , Chazal Geneviève , Carleton Alan , Goridis Christo , Vincent Jean-Didier , Lledo Pierre-Marie
Published: July 2002

Cell adhesion molecules (CAMs) are known to be involved in a variety of developmental processes that play key roles in the establishment of synaptic connectivity during embryonic development, but recent evidence implicates the same molecules in synaptic plasticity of the adult. In the present study, we have used neural CAM (NCAM)-deficient mice, which have learning and behavioral deficits, to evaluate NCAM function in the hippocampal mossy fiber system. Morphological studies demonstrated that fasciculation and laminar growth of mossy fibers were strongly affected, leading to innervation of CA3 pyramidal cells at ectopic sites, whereas individual mossy fiber boutons appeared normal. Electrophysiological recordings performed in hippocampal slice preparations revealed that both basal synaptic transmission and two forms of short-term plasticity, i.e., paired-pulse facilitation and frequency facilitation, were normal in mice lacking all forms of NCAM. However, long-term potentiation of glutamatergic excitatory synapses after brief trains of repetitive stimulation was abolished. Taken together, these results strongly suggest that in the hippocampal mossy fiber system, NCAM is essential both for correct axonal growth and synaptogenesis and for long-term changes in synaptic strength.
Biosynthetic control of molecular weight in the polymerization of the octasaccharide subunits of succinoglycan, a symbiotically important exopolysaccharide of
Rhizobium meliloti
Authors: González Juan E. , Semino Carlos E. , Wang Lai-Xi , Castellano-Torres Laura E. , Walker Graham C.
Published: July 2002

Succinoglycan, a symbiotically important exopolysaccharide of
Rhizobium meliloti
, is composed of polymerized
octasaccharide subunits, each of which consists of one galactose and
seven glucoses with succinyl, acetyl, and pyruvyl modifications.
Production of specific low molecular weight forms of
R.
meliloti
exported and surface polysaccharides, including
succinoglycan, appears to be important for nodule invasion. In a
previous study of the roles of the various
exo
gene
products in succinoglycan biosynthesis,
exoP, exoQ,
and
exoT
mutants were found to synthesize
undecaprenol-linked fully modified succinoglycan octasaccharide
subunits, suggesting possible roles for their gene products in
polymerization or transport. Using improved techniques for analyzing
succinoglycan biosynthesis by these mutants, we have obtained evidence
indicating that
R. meliloti
has genetically separable
systems for the synthesis of high molecular weight succinoglycan and
the synthesis of a specific class of low molecular weight
oligosaccharides consisting of dimers and trimers of the octasaccharide
subunit. Models to account for our unexpected findings are discussed.
Possible roles for the ExoP, ExoQ, and ExoT proteins are compared and
contrasted with roles that have been suggested on the basis of
homologies to key proteins involved in the biosynthesis of O-antigens
and of certain exported or capsular cell surface polysaccharides.
Catalytically inactive lipoprotein lipase expression in muscle of transgenic mice increases very low density lipoprotein uptake: Direct evidence that lipoprotein lipase bridging occurs
in vivo
Authors: Merkel Martin , Kako Yuko , Radner Herbert , Cho Irene S. , Ramasamy Ravi , Brunzell John D. , Goldberg Ira J. , Breslow Jan L.
Published: July 2002

Lipoprotein lipase (LPL) is the central enzyme in plasma triglyceride hydrolysis.
In vitro
studies have shown that LPL also can enhance lipoprotein uptake into cells via pathways that are independent of catalytic activity but require LPL as a molecular bridge between lipoproteins and proteoglycans or receptors. To investigate whether this bridging function occurs
in vivo
, two transgenic mouse lines were established expressing a muscle creatine kinase promoter-driven human LPL (hLPL) minigene mutated in the catalytic triad (Asp
156
to Asn). Mutated hLPL was expressed only in muscle and led to 3,100 and 3,500 ng/ml homodimeric hLPL protein in post-heparin plasma but no hLPL catalytic activity. Less than 5 ng/ml hLPL was found in preheparin plasma, indicating that proteoglycan binding of mutated LPL was not impaired. Expression of inactive LPL did not rescue LPL knock-out mice from neonatal death. On the wild-type (LPL2) background, inactive LPL decreased very low density lipoprotein (VLDL)-triglycerides. On the heterozygote LPL knock-out background (LPL1) background, plasma triglyceride levels were lowered 22 and 33% in the two transgenic lines. After injection of radiolabeled VLDL, increased muscle uptake was observed for triglyceride-derived fatty acids (LPL2, 1.7×; LPL1, 1.8×), core cholesteryl ether (LPL2, 2.3×; LPL1, 2.7×), and apolipoprotein (LPL1, 1.8×; significantly less than cholesteryl ether). Skeletal muscle from transgenic lines had a mitochondriopathy with glycogen accumulation similar to mice expressing active hLPL in muscle. In conclusion, it appears that inactive LPL can act
in vivo
to mediate VLDL removal from plasma and uptake into tissues in which it is expressed.
RNA polymerase of vesicular stomatitis virus specifically associates with translation elongation factor-1 αβγ for its activity
Authors: Das Tapas , Mathur Manjula , Gupta Ashim K. , Janssen George M. C. , Banerjee Amiya K.
Published: July 2002

An RNA-dependent RNA polymerase is packaged within the virions of purified vesicular stomatitis virus, a nonsegmented negative-strand RNA virus, which carries out transcription of the genome RNA into mRNAs both
in vitro
and
in vivo
. The RNA polymerase is composed of two virally encoded polypeptides: a large protein L (240 kDa) and a phosphoprotein P (29 kDa). Recently, we obtained biologically active L protein from insect cells following infection by a recombinant baculovirus expressing L gene. During purification of the L protein from Sf21 cells, we obtained in addition to an active L fraction an inactive fraction that required uninfected insect cell extract to restore its activity. The cellular factors have now been purified, characterized, and shown to be β and γ subunits of the protein synthesis elongation factor EF-1. We also demonstrate that the α subunit of EF-1 remains tightly bound to the L protein in the inactive fraction and βγ subunits associate with the L(α) complex. Further purification of L(α) from the inactive fraction revealed that the complex is partially active and is significantly stimulated by the addition of βγ subunits purified from Sf21 cells. A putative inhibitor(s) appears to co-elute in the inactive fraction that blocked the L(α) activity. The purified virions also package all three subunits of EF-1. These findings have a striking similarity with Qβ RNA phage, which also associates with the bacterial homologue of EF-1 for its replicase function, implicating a possible evolutionary relationship between these host proteins and the RNA-dependent RNA polymerase of RNA viruses.
Repeated pulses of serotonin required for long-term facilitation activate mitogen-activated protein kinase in sensory neurons of
Aplysia
Authors: Michael Dan , Martin Kelsey C. , Seger Rony , Ning Ming-Ming , Baston Rene , Kandel Eric R.
Published: July 2002

Long-term facilitation of the connections between the sensory and motor neurons of the gill-withdrawal reflex in
Aplysia
requires five repeated pulses of serotonin (5-HT). The repeated pulses of 5-HT initiate a cascade of gene activation that leads ultimately to the growth of new synaptic connections. Several genes in this process have been identified, including the transcriptional regulators apCREB-1, apCREB-2, apC/EBP, and the cell adhesion molecule apCAM, which is thought to be involved in the formation of new synaptic connections. Here we report that the transcriptional regulators apCREB-2 and apC/EBP, as well as a peptide derived from the cytoplasmic domain of apCAM, are phosphorylated
in vitro
by
Aplysia
mitogen-activated protein kinase (apMAPK). We have cloned the cDNA encoding apMAPK and show that apMAPK activity is increased in sensory neurons treated with repeated pulses of 5-HT and by the cAMP pathway. These results suggest that apMAPK may participate with cAMP-dependent protein kinase during long-term facilitation in sensory cells by modifying some of the key elements involved in the consolidation of short- to long-lasting changes in synaptic strength.
Unique regulatory properties of the type 2a Ca
2+
channel β subunit caused by palmitoylation
Authors: Qin Ning , Platano Daniela , Olcese Riccardo , Costantin James L. , Stefani Enrico , Birnbaumer Lutz
Published: July 2002

β subunits of voltage-gated Ca
2+
channels are encoded in four genes and display additional molecular diversity because of alternative splicing. At the functional level, all forms are very similar except for β2a, which differs in that it does not support prepulse facilitation of α
1C
Ca
2+
channels, inhibits voltage-induced inactivation of neuronal α
1E
Ca
2+
channels, and is more effective in blocking inhibition of α
1E
channels by G protein-coupled receptors. We show that the distinguishing properties of β2a, rather than interaction with a distinct site of α
1
, are because of the recently described palmitoylation of cysteines in positions three and four, which also occurs in the
Xenopus
oocyte. Essentially, all of the distinguishing features of β2a were lost in a mutant that could not be palmitoylated [β2a(Cys
3,4
Ser)]. Because protein palmitoylation is a dynamic process, these findings point to the possibility that regulation of palmitoylation may contribute to activity-dependent neuronal and synaptic plasticity. Evidence is presented that there may exist as many as three β2 splice variants differing only in their N-termini.
Calcium regulation of a slow post-spike hyperpolarization in vagal afferent neurons
Authors: Cordoba-Rodriguez Ruth , Moore Kimberly A. , Kao Joseph P. Y. , Weinreich Daniel
Published: July 2002

Activation of distinct classes of potassium channels can
dramatically affect the frequency and the pattern of neuronal firing.
In a subpopulation of vagal afferent neurons (nodose ganglion neurons),
the pattern of impulse activity is effectively modulated by a
Ca
2+
-dependent K
+
current. This current
produces a post-spike hyperpolarization (AHP
slow
) that
plays a critical role in the regulation of membrane excitability and is
responsible for spike-frequency accommodation in these neurons.
Inhibition of the AHP
slow
by a number of
endogenous autacoids (e.g., histamine, serotonin,
prostanoids, and bradykinin) results in an increase in the firing
frequency of vagal afferent neurons from <0.1 to >10 Hz. After a
single action potential, the AHP
slow
in nodose neurons
displays a slow rise time to peak (0.3–0.5 s) and a long duration
(3–15 s). The slow kinetics of the AHP
slow
are due, in
part, to Ca
2+
discharge from an intracellular
Ca
2+
-induced Ca
2+
release (CICR) pool. Action
potential-evoked Ca
2+
influx via either L or N type
Ca
2+
channels triggers CICR. Surprisingly, although L type
channels generate 60% of action potential-induced CICR, only
Ca
2+
influx through N type Ca
2+
channels can
trigger the CICR-dependent AHP
slow
. These observations
suggest that a close physical proximity exists between endoplasmic
reticulum ryanodine receptors and plasma membrane N type
Ca
2+
channels and AHP
slow
potassium channels.
Such an anatomical relation might be particularly beneficial for
modulation of spike-frequency adaptation in vagal afferent neurons.
Analysis of human peripheral blood T cells and single-cell-derived T cell clones uncovers extensive clonal CpG island methylation heterogeneity throughout the genome
Authors: Zhu Xiaoxiang , Deng Chun , Kuick Rork , Yung Raymond , Lamb Barbara , Neel James V. , Richardson Bruce , Hanash Samir
Published: July 2002

Methylation of cytosine residues in CpG dinucleotides is generally associated with silencing of gene expression. DNA methylation, as a somatic event, has the potential of diversifying gene expression in individual cells of the same lineage. There is little quantitative data available concerning the extent of methylation heterogeneity in individual cells across the genome. T cells from the peripheral blood can be grown as single-cell-derived clones and can be analyzed with respect to their DNA methylation patterns by restriction landmark genomic scanning. The use of the methylation-sensitive enzymeNotI to cut and end-label DNA fragments before their separation in two dimensions provides a quantitative assessment of methylation atNotI sites that characteristically occur in CpG islands. We have undertaken quantitative analysis of two-dimensional DNA patterns to determine the extent of methylation heterogeneity atNotI sites between peripheral blood single-cell-derived T cell clones from the same individual. A total of 1,068NotI-tagged fragments were analyzed. A subset of 156 fragments exhibited marked methylation heterogeneity atNotI sites between clones. Their average intensity among clones correlated with their intensity in uncultured, whole-blood-derived T cells, indicating that the methylation heterogeneity observed in clones was largely attributable to methylation heterogeneity between the individual cells from which the clones were derived. We have cloned one fragment that exhibited variableNotI-site methylation between clones. This fragment contained a novel CpG island for a gene that we mapped to chromosome 4. The methylation status of theNotI site of this fragment correlated with expression of the corresponding gene. Our data suggest extensive diversityin vivoin the methylation and expression profiles of individual T cells at multiple unrelated loci across the genome.
A catalytic switch and the conversion of streptokinase to a fibrin-targeted plasminogen activator
Authors: Reed Guy L. , Houng Aiilyan K. , Liu Lin , Parhami-Seren Behnaz , Matsueda Lee H. , Wang Shunguang , Hedstrom Lizbeth
Published: July 2002

Plasminogen (Pg) activators such as streptokinase (SK) save lives by generating plasmin to dissolve blood clots. Some believe that the unique ability of SK to activate Pg in the absence of fibrin limits its therapeutic utility. We have found that SK contains an unusual NH2-terminal “catalytic switch” that allows Pg activation through both fibrin-independent and fibrin-dependent mechanisms. Unlike SK, a mutant (rSKΔ59) fusion protein lacking the 59 NH2-terminal residues was no longer capable of fibrin-independent Pg activation (kcat/Kmdecreased by >600-fold). This activity was restored by coincubation with equimolar amounts of the NH2-terminal peptide rSK1–59. Deletion of the NH2terminus made rSKΔ59 a Pg activator that requires fibrin, but not fibrinogen, for efficient catalytic function. The fibrin-dependence of the rSKΔ59 activator complex apparently resulted from selective catalytic processing of fibrin-bound Pg substrates in preference to other Pg forms. Consistent with these observations, the presence (rSK) or absence (rSKΔ59) of the SK NH2-terminal peptide markedly altered fibrinolysis of human clots suspended in plasma. Like native SK, rSK produced incomplete clot lysis and complete destruction of plasma fibrinogen; in contrast, rSKΔ59 produced total clot lysis and minimal fibrinogen degradation. These studies indicate that structural elements in the NH2terminus are responsible for SK’s unique mechanism of fibrin-independent Pg activation. Because deletion of the NH2terminus alters SK’s mechanism of action and targets Pg activation to fibrin, there is the potential to improve SK’s therapeutic efficacy.
SLAP, a dimeric adapter protein, plays a functional role in T cell receptor signaling
Authors: Tang Jie , Sawasdikosol Sansana , Chang Jin-Hong , Burakoff Steven J.
Published: July 2002

Engagement of the T cell antigen receptor (TCR) leads to rapid activation of protein tyrosine kinases, which in turn phosphorylate downstream enzymes and adapter proteins. Some adapter proteins, such as SLP-76, Vav, and LAT, positively regulate TCR-mediated signal transduction, whereas others, such as Cbl, play an inhibitory role. SLAP (Src-like adapter protein), an adapter protein containing a Src homology 3 and a Src homology 2 domain, was isolated from a yeast interacting screen by using N-terminal Cbl as bait. N-terminal Cbl interacts with SLAP
in vivo
and
in vitro
in a tyrosine phosphorylation-independent manner. We observed that SLAP is expressed in T cells, and upon TCR activation, SLAP interacts with ZAP-70, Syk, LAT, and TCRζ chain in Jurkat T cells. In transiently transfected COS-7 cells, SLAP forms separate complexes with ZAP-70, Syk, and LAT through its Src homology 2 domain. Overexpression of a C-terminal-truncated SLAP mutant down-regulates nuclear factor of activated T cells-AP1 activity. We have evidence that SLAP forms homodimers through its C-terminal region. Serial truncations and mutations in the C terminus of SLAP demonstrate that there is a correlation between the loss of dimerization and the inhibition of nuclear factor of activated T cells-AP1 activity. The
in vivo
association of SLAP with key signaling molecules and its inhibition of T cell activation suggests that SLAP plays an important role in TCR-mediated signal transduction.