bluuebunny/crossref_metadata_2025 huggingface dataset:
High-affinity binding sites for related fibroblast growth factor ligands reside within different receptor immunoglobulin-like domains.
Authors: Cheon H G , LaRochelle W J , Bottaro D P , Burgess W H , Aaronson S A
Published: May 2006

Growth factors of the fibroblast growth factor (FGF) family bind receptors whose external domains are organized in a series of immunoglobulin-like loops. We engineered expression constructs in which cDNAs encoding individual immunoglobulin-like domains of the keratinocyte growth factor (KGF/FGF-7) receptor were fused to the mouse immunoglobulin heavy chain Fc domain (HFc). Each chimera was efficiently secreted from NIH 3T3 transfectants and migrated at the predicted molecular mass after SDS/PAGE. Scatchard analysis revealed that the chimera containing immunoglobulin-like domains 2 (D2) and 3 (D3) bound KGF and acidic FGF at high affinities comparable to the native receptor. However, individual immunoglobulin-like domain chimeras demonstrated marked specificity in their ligand interactions. D2-HFc bound acidic FGF at high affinity, whereas it did not detectably interact with KGF. Conversely, D3-HFc bound KGF at high affinity but exhibited no detectable interaction with acidic FGF. Their selective ligand binding properties were confirmed by the specific neutralization of acidic FGF or KGF mitogenic activity using D2 or D3 HFc, respectively. All of these findings establish that the major binding sites for related FGF ligands are localized to distinct receptor immunoglobulin-like domains.
Endothelial cells are activated by cytokine treatment to kill an intravascular parasite, Schistosoma mansoni, through the production of nitric oxide.
Authors: Oswald I P , Eltoum I , Wynn T A , Schwartz B , Caspar P , Paulin D , Sher A , James S L
Published: May 2006

Like many pathogens that undergo an intravascular stage of development, larvae of the helminth parasite Schistosoma mansoni migrate through the blood vessels, where they are in close contact with endothelial cells. In vitro exposure of murine endothelial cells to various cytokines (interferon gamma, tumor necrosis factor alpha, and interleukin 1 alpha or 1 beta) resulted in their activation to kill schistosomula through an arginine-dependent mechanism involving production of nitric oxide (NO). Cytokine-treated endothelial cells showed increased expression of mRNA for the inducible form of the NO synthase, and both NO production and larval killing were suppressed by treatment with competitive inhibitors. The effector function of cytokine-treated endothelial cells was similar to that of activated inflammatory tissue macrophages, although activation appeared to be differentially regulated in these two cell types. Activated endothelial cells killed older (18-day) forms of the parasite, such as those currently thought to be a primary target of immune elimination in the lungs of mice previously vaccinated with radiation-attenuated cercariae, as well as newly transformed larvae. In C57BL/6 mice, which become resistant to S. mansoni infection as a result of vaccination with irradiated cercariae, endothelial cell morphology characteristic of activation was observed in the lung by 1-2 weeks after challenge infection. Similar endothelial cell changes were absent in P-strain mice, which do not become resistant as a result of vaccination. Together, these observations indicate that endothelial cells, not traditionally considered to be part of the immune system, may play an important role in immunity to S. mansoni and, by means of NO-dependent killing, could serve as effectors of resistance to other intravascular pathogens.
Thallium counterion distribution in cubic insulincrystals determined from anomalous x-ray diffraction data.
Authors: Badger J , Li Y , Caspar D L
Published: May 2006

To determine the distribution of monovalent
cations around a protein we have measured anomalous scattering diffraction data
from Tl-containing cubic insulin crystals at pH 8 and pH 10.5. The differences
between Bijvoet reflection pairs within each set of data were used to calculate
anomalous scattering difference maps. Both maps show the same six Tl+ sites,
which include two well-ordered Tl+ ions previously identified from isomorphous
exchange experiments. The other four sites constitute a second class of cations,
which, while much more mobile than the protein atoms, are associated with
particular ligating groups. Three of the six Tl+ sites are created exclusively
by protein main and side chain carbonyl dipoles rather than negatively charged
groups. All of the Tl+ ions are positioned so as to interact with both protein
atoms and water molecules. The Tl+ occupancies appear to depend in a complex way
on interactions with each other and flexibility in the protein structure. The
combined occupancies of these cations are slightly less than is required to
neutralize the net protein charge of approximately -2e at pH 8 but account for
only about half of the approximately -5e protein charge at pH 10.5. Thus, more
disordered counterions, not seen in these Bijvoet anomalous scattering
difference maps, are more numerous at higher protein net charge.
Time-dependent diffusion of water in a biologicalmodel system.
Authors: Latour L L , Svoboda K , Mitra P P , Sotak C H
Published: May 2006

Packed erythrocytes are ideally suited as a model
system for the study of water diffusion in biological tissue, because cell size,
membrane permeability, and extracellular volume fraction can be varied
independently. We used a pulsed-field-gradient spin echo NMR technique to
measure the time-dependent diffusion coefficient D(t) in packed erythrocytes.
The long-time diffusion constant, D(eff), depends sensitively on the
extracellular volume fraction. This may explain the drop in D(eff) during the
early stages of brain ischemia, where just minutes after an ischemic insult the
extra-cellular volume in the affected region of the brain is significantly
reduced. Using an effective medium formula, we estimate the erythrocyte membrane
permeability, in good agreement with measurements on isolated cells. From the
short-time behavior of D(t), we determine the surface-to-volume ratio of the
cells, approximately (0.72 micron)-1.
Vaccination of chimpanzees against infection by thehepatitis C virus.
Authors: Choo Q L , Kuo G , Ralston R , Weiner A , Chien D , Van Nest G , Han J , Berger K , Thudium K , Kuo C
Published: May 2006

A high incidence of community-acquired hepatitis C
virus infection that can lead to the progressive development of chronic active
hepatitis, liver cirrhosis, and primary hepatocellular carcinoma occurs
throughout the world. A vaccine to control the spread of this agent that
represents a major cause of chronic liver disease is therefore needed. Seven
chimpanzees (Pan troglodytes) have been immunized with both putative envelope
glycoproteins [E1 (gp33) and E2 (gp72)] that were copurified from HeLa cells
infected with a recombinant vaccinia virus expression vector. Despite the
induction of a weak humoral immune response to these viral glycoproteins in
experimentally infected chimpanzees, a strong humoral immune response was
obtained in all vaccines. The five highest responders showed complete protection
against an i.v. challenge with homologous hepatitis C virus 1. The remaining two
vaccines became infected, but both infection and disease may have been
ameliorated in comparison with four similarly challenged control chimpanzees,
all of which developed acute hepatitis and chronic infections. These results
provide considerable encouragement for the eventual control of hepatitis C virus
infection by vaccination.
NADPH: ferredoxin oxidoreductase acts as a paraquatdiaphorase and is a member of the soxRS regulon.
Authors: Liochev S I , Hausladen A , Beyer W F , Fridovich I
Published: May 2006

Soluble extracts of Escherichia coli contain four
NADPH:paraquat diaphorases that were separable by anion-exchange HPLC over Mono
Q. One of these was induced when the cells were exposed to paraquat. This was
the case in a soxRS-competent strain but not in a soxRS-null strain, while a
soxRS-constitutive strain overexpressed this diaphorase without the stimulus of
exposure to paraquat. This NADPH:paraquat diaphorase could use cytochrome c or
nitroblue tetrazolium as an electron acceptor, whereas O2 was a relatively poor
acceptor. This diaphorase was identified as the NADPH:ferredoxin reductase. A
role for reduced ferredoxin and flavodoxin in the adaptive soxRS response to
oxidative stress and in the regulation of the redox status of soxR is
discussed.
P nucleotide insertions and the resolution of hairpinDNA structures in mammalian cells.
Authors: Lewis S M
Published: May 2006

Two lines of evidence point to a hairpin DNA
intermediate in V(D)J joining (V, variable; D, diversity; J, joining) [Lieber,
M.R. (1991) FASEB J. 4, 2934-2944]. One is the presence of P nucleotide
insertions (short inverted-repeat sequence) in V(D)J junctions [Lafaille, J. J.,
DeCloux, A., Bonneville, M., Takagaki, Y. & Tonegawa, S. (1989) Cell 59,
859-870]; a second is the detection of site-specifically broken DNA molecules
with covalently closed (hairpin) termini in thymus DNA [Roth, D. B., Menetski,
J. P., Nakajima, P., Bosma, M. J. & Gellert, M. (1989) Cell 70, 983-991].
However, P nucleotide insertions could be generated in ways not involving a
hairpin structure, and because physical evidence for hairpin-ended DNA fragments
has been obtained only with mutant mice, there is some uncertainty regarding the
role of hairpin molecules in the normal V(D)J joining pathway. To determine
whether mammalian cells are capable of metabolizing this odd type of DNA
terminus and whether, in doing so, junctions with P insertions are in fact
created, a linear DNA molecule with a hairpin closure at each end was
transfected into several murine cell lines. The hairpin-ended molecules were
recircularized, and the junctions exhibited P insertions at a high frequency.
This result directly links the presence of P insertions to a hairpin precursor,
providing strong evidence for the notion that a hairpin DNA intermediate exists
in V(D)J recombination. A comparison of hairpin end joining in various cells,
including those derived from mice with the severe combined immunodeficiency
(scid) mutation, is presented.
Trimethylpsoralen induces small deletion mutations inCaenorhabditis elegans.
Authors: Yandell M D , Edgar L G , Wood W B
Published: May 2006

To examine the mutagenic spectrum of
4,5',8-trimethylpsoralen (TMP) in Caenorhabditis elegans, we isolated
mutations in the unc-22 and pal-1 genes following TMP mutagenesis and analyzed
them for restriction fragment length polymorphisms by Southern blot. Eleven of
21 unc-22 mutations exhibited restriction fragment length polymorphisms, 8 of
which were deletions of between 0.10 and 15 kb in length. Both of two pal-1
mutations were also small deletions within this size range. Comparison of our
results with previous studies on mutagenesis by gamma-rays and x-rays suggests
that the mutagenic spectrum of TMP may be similar. TMP should be useful in
generating mutations that cause complete loss of function of single genes and
that are likely to result in allele-specific DNA polymorphisms.
Recombinant human respiratory syncytial virus (RSV)monoclonal antibody Fab is effective therapeutically when introduced directlyinto the lungs of RSV-infected mice.
Authors: Crowe J E , Murphy B R , Chanock R M , Williamson R A , Barbas C F , Burton D R
Published: May 2006

Previously, recombinant human respiratory
syncytial virus (RSV) monoclonal antibody Fabs were generated by antigen
selection from random combinatorial libraries displayed at the tip of
filamentous phage. Two such Fabs, which exhibited high binding affinity for RSV
F glycoprotein (a major protective antigen), were evaluated for therapeutic
efficacy in infected mice just before or at the time of peak virus replication
in the lungs. Fab 19, which neutralized RSV infectivity with high efficiency in
tissue culture, was effective therapeutically when delivered directly into the
lungs by intranasal instillation under anesthesia. In contrast, RSV Fab 126,
which failed to neutralize virus in cell culture, did not exhibit a therapeutic
effect under these conditions. The amount of Fab 19 required to effect a 5000-
to 12,000-fold reduction in titer of RSV in the lungs within 24 hr was rather
small. In four separate experiments, a single instillation of 12.9-50 micrograms
of RSV Fab 19 was sufficient to achieve such a reduction in pulmonary virus in a
25g mouse. The use of Fabs instead of the whole immunoglobulin molecules from
which they are derived reduced the protein content of a therapeutic dose. This
is important because the protein load that can be delivered effectively into the
lungs is limited. The therapeutic effect of a single treatment with Fab 19 was
not sustained, so that a rebound in pulmonary virus titer occurred on the 2nd
day after treatment. This rebound in pulmonary RSV titer could be prevented by
treating infected mice with a single dose of Fab 19 daily for 3 days. These
observations suggest that human monoclonal Fabs grown in Escherichia coli may
prove useful in the treatment of serious RSV disease as well as diseases caused
by other viruses where replication in vivo is limited primarily to the lumenal
lining of the respiratory tract.
Inherited resistance to activated protein C iscorrected by anticoagulant cofactor activity found to be a property of factorV.
Authors: Dahlbäck B , Hildebrand B
Published: May 2006

Recently, our laboratory described a defect in
anticoagulant response to activated protein C (APC). This response, APC
resistance, was shown to be inherited and associated with familial
thrombophilia. As other possible mechanisms were excluded, APC resistance was
hypothesized to be due to deficiency of a previously unrecognized cofactor of
APC. The aim of the present study was to isolate and characterize this factor.
Plasma from an individual with pronounced inherited APC resistance was used as
test plasma in a biological assay which monitored APC cofactor activity during
its isolation from normal plasma. A purification procedure was devised that
yielded a protein which was shown to be identical to coagulation factor V. It
proved impossible to separate the APC cofactor activity from factor V, even by
affinity chromatography using a monoclonal antibody against factor V. The
affinity-purified factor V corrected the poor anticoagulant response to APC of
APC-resistant plasma in a dose-dependent manner. Because the APC-resistant
plasma contained normal levels of factor V procoagulant activity, the results
indicated APC resistance to be due to a selective defect in the anticoagulant
function of factor V. The present results show factor V not only to express
procoagulant properties after its activation by thrombin but also to play an
important part in the anticoagulant system as cofactor to APC.
Identification of an ion channel-forming motif in theprimary structure of CFTR, the cystic fibrosis chloride channel.
Authors: Oblatt-Montal M , Reddy G L , Iwamoto T , Tomich J M , Montal M
Published: May 2006

Synthetic peptides with sequences representing
putative transmembrane (M) segments of CFTR (the cystic fibrosis transmembrane
conductance regulator) were used as tools to identify the involvement of such
segments in forming the ionic pore of the CFTR Cl- channel. Peptides with
sequences corresponding to M2 and M6 form anion-selective channels after
reconstitution in lipid bilayers. In contrast, peptides with the sequences of
M1, M3, M4, and M5, or peptides of the same amino acid composition as M2 and M6
but with scrambled sequences, do not form channels. Conductive heterooligomers
of M2 and M6 exhibit a single channel conductance of 8 pS (in 0.15 M KCl) and a
95% selectivity for anions over cations, properties that emulate both the
conductance and the selectivity of the authentic CFTR channel. The
identification of sequence-specific motifs that account for key functional
attributes of the CFTR channel suggests that such modules may represent
fundamental units of function and are plausible constituents of the pore-forming
structure of the CFTR Cl- channel.
Efficient removal of uracil from G.U mispairs by themismatch-specific thymine DNA glycosylase from HeLa cells.
Authors: Neddermann P , Jiricny J
Published: May 2006

The uracil DNA glycosylases (EC 3.2.2.3)
characterized to date remove uracil from DNA irrespective of whether it is base
paired with adenine or mispaired with guanine in double-stranded substrates or
whether it is found in single-stranded DNA. We report here the characterization
of uracil glycosylase activity that can remove the base solely from a mispair
with guanine. It does not recognize uracil either in A.U pairs or in
single-stranded substrates. The enzyme, a 55-kDa polypeptide, was previously
characterized as a mismatch-specific thymine DNA glycosylase and was thought to
be responsible solely for the correction (to G.C) of G.T mispairs that arise as
a result of spontaneous hydrolytic deamination of 5-methylcytosine to thymine.
Given the broader substrate specificity of the enzyme (in addition to uracil and
thymine, the protein can also remove 5-bromouracil from mispairs with guanine),
we propose that its biological role in vivo may also include the correction of a
subset of G.U mispairs inefficiently removed by the more abundant ubiquitous
uracil glycosylases, such as those arising from cytosine deamination in G+C-rich
regions of the genome.
Nitric oxide: a mediator in rat tubularhypoxia/reoxygenation injury.
Authors: Yu L , Gengaro P E , Niederberger M , Burke T J , Schrier R W
Published: May 2006

Nitric oxide (NO), among several other functions,
may play a role in hypoxia and reoxygenation injury due to its free radical
nature and high reactivity with the superoxide radical to yield peroxynitrite,
an oxidant molecule. The present study was undertaken to evaluate a potential
role for NO, either endogenous or exogenous, in a model of hypoxia/reoxygenation
(H/R) in freshly isolated rat proximal tubules. NO synthase activity, as
assessed by conversion of L-[3H]arginine to L-[3H]citrulline, was detected in
normoxic tubules. This activity could be inhibited by N-nitro-L-arginine methyl
ester (L-NAME), a NO synthase inhibitor, and was stimulated by 15 min of
hypoxia. The injury in proximal tubules caused by 15 min of hypoxia followed by
35 min of reoxygenation was completely prevented by L-NAME as assessed by
release of lactate dehydrogenase, whereas D-NAME, which does not inhibit NO
synthase, had no effect. In contrast, L-arginine (NO substrate) enhanced the H/R
injury. These effects were paralleled by nitrite/nitrate production. In separate
experiments, the addition of sodium nitroprusside, a NO donor, to proximal
tubules enhanced the H/R injury; this effect could be blocked by hemoglobin, a
NO scavenger. Also, addition of nitroprusside reversed L-NAME protection against
H/R injury. These results demonstrate that NO is synthesized in rat proximal
tubules and participates as one of the mediators in rat tubular H/R
injury.
Direct demonstration of three different states of thepancreatic cholecystokinin receptor.
Authors: Talkad V D , Fortune K P , Pollo D A , Shah G N , Wank S A , Gardner J D
Published: May 2006

We used rat pancreatic acini as well as COS-7
cells transfected with the cloned pancreatic cholecystokinin (CCK) receptor and
measured the abilities of CCK octapeptide (CCK-8) and L-364,718 (a CCK receptor
antagonist) to inhibit binding of 125I-labeled CCK-8 (125I-CCK-8) and
[3H]L-364,718. With pancreatic acini 125I-CCK-8 bound to two different states of
the CCK receptor. The high-affinity state (1% of the receptors) had a Kd for
CCK-8 of 985 pM and the low-affinity state (19% of the receptors) had a Kd for
CCK-8 of 30 nM. [3H]L-364,718 bound to low-affinity receptors and to a
previously unrecognized very-low-affinity state (80% of the receptors) having a
Kd for CCK-8 of 13 microM. L-364,718 had the same affinity (Kd 3 nM) for each of
the three different states of the CCK receptor. Similar measurements using
transfected COS cells also identified three different states of the CCK
receptor, with the very-low-affinity state being the most abundant. Thus, the
ability of the CCK receptor to exist in three different states is an intrinsic
property of the CCK receptor molecule itself.
Nuclear matrix proteins in human coloncancer.
Authors: Keesee S K , Meneghini M D , Szaro R P , Wu Y J
Published: May 2006

The nuclear matrix is the nonchromatin scaffolding
of the nucleus. This structure confers nuclear shape, organizes chromatin, and
appears to contain important regulatory proteins. Tissue specific nuclear matrix
proteins have been found in the rat, mouse, and human. In this study we compared
high-resolution two-dimensional gel electropherograms of nuclear matrix protein
patterns found in human colon tumors with those from normal colon epithelia.
Tumors were obtained from 18 patients undergoing partial colectomy for
adenocarcinoma of the colon and compared with tissue from 10 normal colons. We
have identified at least six proteins which were present in 18 of 18 colon
tumors and 0 of 10 normal tissues, as well as four proteins present in 0 of 18
tumors and in 10 of 10 normal tissues. These data, which corroborate similar
findings of cancer-specific nuclear matrix proteins in prostate and breast,
suggest that nuclear matrix proteins may serve as important markers for at least
some types of cancer.
Differential roles of the transposon termini in IS91transposition.
Authors: Mendiola M V , Bernales I , de la Cruz F
Published: May 2006

Insertion sequence 91 (IS91) inserts specifically
at GTTC or CTTG target sequences without duplication of the target. After
insertion, the right inverted repeat (IRR) lies adjacent to the 3' end of
the target sequences (or 5' to the complementary sequence CAAG or GAAC). We
have analyzed the effects of alteration of each terminus of IS91 on
transposition activity in Escherichia coli. IRR is absolutely required for
transposition. Deletion analysis indicates that a 14-bp segment is not
sufficient, but an 81-bp sequence within the IRR region is sufficient.
Furthermore, the GTTC/CTTG target site is also required. The left inverted
repeat (IRL) of IS91 is dispensable. Plasmid fusions originated by one-ended
transposition of IS91 derivatives lacking IRL occur at about the same frequency
as cointegrate formation observed for the wild-type element. In the
one-ended-type fusions, the inserted fragment of donor DNA is flanked at one end
(constant end) by IRR and at the other end by a GTTC or CTTG sequence present in
the donor (variable end) in a way that usually results in multiple tandem
insertions of the donor plasmid in the target site. These results are easily
accommodated by a rolling-circle replicative transposition mechanism. This model
also draws support from the finding that the IS91 transposase is related in
sequence to the superfamily of rolling-circle replication proteins and the
observation that IRR shows some conservation in sequence and secondary structure
with the origins of replication of some rolling-circle replication
plasmids.
Neuroanatomical correlates of encoding in episodic memory: levels of processing effect.
Authors: Kapur S , Craik F I , Tulving E , Wilson A A , Houle S , Brown G M
Published: May 2006

Cognitive studies of memory processes demonstrate that memory for stimuli is a function of how they are encoded; stimuli processed semantically are better remembered than those processed in a perceptual or shallow fashion. This study investigates the neural correlates of this cognitive phenomenon. Twelve subjects performed two different cognitive tasks on a series of visually presented nouns. In one task, subjects detected the presence or absence of the letter a; in the other, subjects categorized each noun as living or nonliving. Positron emission tomography (PET) scans using 15O-labeled water were obtained during both tasks. Subjects showed substantially better recognition memory for nouns seen in the living/nonliving task, compared to nouns seen in the a-checking task. Comparison of the PET images between the two cognitive tasks revealed a significant activation in the left inferior prefrontal cortex (Brodmann's areas 45, 46, 47, and 10) in the semantic task as compared to the perceptual task. We propose that memory processes are subserved by a wide neurocognitive network and that encoding processes involve preferential activation of the structures in the left inferior prefrontal cortex.
Mutational analysis of a critical signaling domain of the human interleukin 4 receptor.
Authors: Seldin D C , Leder P
Published: May 2006

The human interleukin 4 receptor (hIL-4R) is a member of a superfamily of cytokine receptors defined by conserved features of their extracellular domains. The intracellular domains of the hIL-4R and of other members of this family lack any recognizable enzymatic motifs, though ligand-dependent tyrosine phosphorylation of these receptors has been observed. Recent studies have suggested that serine-rich and acidic domains within the cytoplasmic portions of cytokine receptors might be required for signal transduction. Using deletion and truncation mutants of the hIL-4R, we have explored an essential 39-amino acid signaling domain that is rich in acidic amino acid residues and in serine residues that form consensus phosphorylation sites for known serine/threonine kinases. To assess the contribution of these motifs to signaling, we engineered site-directed mutants of these residues. Surprisingly, cells expressing mutant hIL-4R lacking either the serine or the acidic amino acids retain the ability of cells expressing the wild-type receptor to proliferate in hIL-4. Furthermore, receptors in which all six cytoplasmic tyrosines are absent can function, suggesting that tyrosine phosphorylation of the receptor may be an epiphenomenon rather than a requisite event in signaling.
Molecular cloning and functional expression of the bumetanide-sensitive Na-K-Cl cotransporter.
Authors: Xu J C , Lytle C , Zhu T T , Payne J A , Benz E , Forbush B
Published: May 2006

By mediating the coupled movement of Na, K, and Cl ions across the plasma membrane of most animal cells, the bumetanide-sensitive Na-K-Cl cotransporter (NKCC) plays a vital role in the regulation of ionic balance and cell volume. The transporter is a central element in the process of vectorial salt transport in secretory and absorptive epithelia. A cDNA encoding a Na-K-Cl cotransport protein was isolated from a shark rectal gland library by screening with monoclonal antibodies to the native shark cotransporter. The 1191-residue protein predicted from the cDNA sequence has 12 putative transmembrane domains flanked by large cytoplasmic N and C termini. Regulatory phosphoacceptor residues in isolated peptides are identified as Thr-189 and Thr-1114 in the predicted sequence. Northern blot analysis identified a 7.4-kb mRNA in rectal gland and most other shark tissues; a 5.2-kb mRNA was restricted to shark kidney. Homology with an uncharacterized gene from Caenorhabditis elegans and with the thiazide-sensitive Na-Cl cotransporter of flounder urinary bladder was found over most of the coding region; shorter stretches of homology were found with a C. elegans cDNA and with an uncharacterized gene of cyanobacterium. Human HEK-293 cells have been stably transfected with the shark cDNA and shown to express Na-K-Cl cotransport activity with the bumetanide sensitivity of the shark protein. The expressed transporter is functionally quiescent in the host cells and can be activated by depleting the cells of chloride.
Cloning of a 3-methyladenine-DNA glycosylase from Arabidopsis thaliana.
Authors: Santerre A , Britt A B
Published: May 2006

We have isolated an Arabidopsis thaliana cDNA that complements the methyl methanesulfonate-sensitive phenotype of an Escherichia coli double mutant deficient in 3-methyladenine glycosylases (DNA-3-methyladenine glycosidases I and II, EC 3.2.2.20 and 3.2.2.21, respectively, encoded by tag and alkA). Expression of the Arabidopsis cDNA enhances the methyl methanesulfonate resistance of the E. coli double mutant by nearly four orders of magnitude. The cDNA corresponds to a single-copy, nuclearly encoded sequence which specifies a predicted 28.1-kDa protein with a charge of +8 at pH 7.0. Enzymatic analysis of extracts prepared from the transformed mutants indicates that the cDNA encodes a 3-methyladenine glycosylase. The predicted amino acid sequence of the Arabidopsis glycosylase has significant homology to other eukaryotic 3-methyladenine glycosylases.
4,5-Dianilinophthalimide: a protein-tyrosine kinase inhibitor with selectivity for the epidermal growth factor receptor signal transduction pathway and potent in vivo antitumor activity.
Authors: Buchdunger E , Trinks U , Mett H , Regenass U , Müller M , Meyer T , McGlynn E , Pinna L A , Traxler P , Lydon N B
Published: May 2006

Deregulated signal transduction via the epidermal growth factor receptor (EGF-R) family of protein-tyrosine kinase growth factor receptors is associated with proliferative diseases. We describe a class of compounds (4,5-dianilinophthalimides) that inhibit the EGF-R protein-tyrosine kinase in vitro with high selectivity. In cells, 4,5-dianilinophthalmide selectively inhibited both ligand-induced EGF-R and p185c-erbB2 autophosphorylation and c-fos mRNA induction. Antitumor activity could be demonstrated in vivo against xenografts of the A431 and SK-OV-3 tumors, which overexpress the EGF-R and p185c-erbB2, respectively. In contrast, a platelet-derived growth factor-driven tumor was not inhibited by 4,5-dianilinophthalimide, which is compatible with its cellular selectivity and hypothesized mechanism of action. No overt cumulative toxicity was observed during treatment even though high efficacy was observed, indicating a good therapeutic window. 4,5-Dianilinophthalimides may offer therapeutic agents for the treatment of hyperproliferative diseases that overexpress EGF-R family protein-tyrosine kinases or their ligands.
Interplay of positive and negative effectors in function of the C-terminal repeat domain of RNA polymerase II.
Authors: Li Y , Kornberg R D
Published: May 2006

RNA polymerase II lacking a C-terminal domain (CTD) was active in transcription with purified proteins from yeast but failed to support transcription in a yeast extract. CTD dependence could be reconstituted in the purified system by addition of two fractions from the extract. An inhibitory fraction abolished transcription by both wild-type and CTD-less RNA polymerases; a stimulatory fraction restored activity of the wild-type polymerase but had a much lesser effect on the CTD-less enzyme. Parallel results were obtained with the use of a kinase inhibitor that prevents phosphorylation of the CTD by RNA polymerase II initiation factor b. The kinase inhibitor abolished transcription by wild-type polymerase in yeast extract but had no significant effect in the purified system. The requirement for both the CTD and kinase action for transcription in an extract indicates that CTD phosphorylation is involved in opposing the negative effector in the extract. Factor b must play a role(s) in addition to phosphorylation of the CTD because it was still required for transcription with polymerase lacking a CTD in the purified system.
Working parents: the impact of day care and breast-feeding on cytomegalovirus infections in offspring.
Authors: Stagno S , Cloud G A
Published: May 2006

The increase in the popularity of breast feeding and utilization of child care arrangements are having a major effect on the epidemiology of cytomegalovirus infections. The impact is greater for women of upper socioeconomic background who send their toddlers to day-care centers and for day-care workers. If primary cytomegalovirus infections occur during pregnancy it is logical to anticipate more cases of severe congenital infections.
Genital human papillomavirus infection.
Authors: Lowy D R , Kirnbauer R , Schiller J T
Published: May 2006

Genital human papillomavirus (HPV) infection is a common sexually transmitted disease that at the present time is not effectively controlled or treated. Many infections are inapparent and transient. However, some HPV infections result in persistent lesions that in some cases undergo carcinogenic progression. A subset of genital HPVs, designated high-risk types, are preferentially associated with high-grade dysplasias and carcinomas. About 90% of cervical cancers contain high-risk HPV DNA, most often HPV16. Development of a subunit vaccine against high-risk genital HPVs is a desirable and, it appears, an increasingly feasible long-term goal. The viral E6 and E7 oncoproteins are selectively maintained and expressed in progressed HPV tumors and could potentially be targets for therapeutic vaccines. The L1 major virion structural proteins have recently been shown to self-assemble into virus-like particles when expressed in insect cells. These particles might serve as the basis for a prophylactic vaccine to prevent genital HPV infection.
Formation of 4-hydroxy-2-nonenal-modified proteins in the renal proximal tubules of rats treated with a renal carcinogen, ferric nitrilotriacetate.
Authors: Toyokuni S , Uchida K , Okamoto K , Hattori-Nakakuki Y , Hiai H , Stadtman E R
Published: May 2006

An iron chelate, ferric nitrilotriacetate (Fe-NTA), induces proximal tubular necrosis, a consequence of lipid peroxidation, that finally leads to a high incidence of renal adenocarcinoma in rodents. Lipid peroxidation as monitored by formation of thiobarbituric acid-reactive substances and free 4-hydroxy-2-nonenal (HNE) was observed in the kidney homogenates of rats treated with Fe-NTA. Based on the fact that HNE is capable of reacting with cellular proteins, we attempted to detect the localization of HNE-modified proteins in rat kidney tissues with an immunohistochemical procedure. By means of an immunohistochemical technique using polyclonal antibody against the HNE-modified proteins, it was shown that HNE-modified proteins are formed in the target cells of this carcinogenesis model. HNE-modified proteins were detected in the renal proximal tubules 1 hr after i.p. administration of Fe-NTA (15 mg of iron per kg). Intense positivity was found in the cells with degeneration. After 6 hr, the level of HNE-protein conjugates decreased due to the subsequent necrosis. The intensity of the immunochemical reaction with HNE-modified proteins increased in parallel with an increase in the amounts of thiobartituric acid-reactive substances and free HNE that were found. Furthermore, histochemical detection of aldehydes by cold Schiff's reagent demonstrated that location of aldehydes was identical to that of the HNE-modified proteins determined by immunohistochemical procedures. It would thus appear that the production of HNE, a genotoxic and mutagenic aldehyde, and its reaction with proteins may play a role in Fe-NTA-induced renal carcinogenesis.
Fib420: a normal human variant of fibrinogen with two extended alpha chains.
Authors: Fu Y , Grieninger G
Published: May 2006

In fibrinogen, alpha E chains form a subpopulation of alpha subunits that are distinguished by a carboxyl extension homologous to the C termini of the other two constituent chains: beta and gamma. The molecular mass of alpha E is > 50% greater than that of the common alpha subunit, due in part to an extra 236 amino acids. These residues are encoded by exon VI, a recently discovered extension of the fibrinogen alpha gene. Additional mass is contributed by posttranslational processing, including N-glycosylation, which, based on experiments with the inhibitor tunicamycin, was found to account in large measure for alpha E migration on SDS/PAGE at approximately 110 kDa rather than at its calculated mass of 92,843 Da. An antibody specific for the exon VI-encoded domain of alpha E (anti-VI) and capable of recognizing alpha E-containing fibrinogen in both native and denatured form was generated using a recombinant protein as immunogen. Its use in Western blot analysis of fractions of normal human blood (plasma and preparations of fibrinogen) revealed a single, sharp, alpha E-containing band migrating behind the position of the broad, predominant fibrinogen band, (alpha beta gamma)2. Designation of the upper band as Fib420, an approximately 420-kDa homodimer of the formula (alpha E beta gamma)2, is based on the overwhelming proportion of alpha E subunits (> 80% of the total alpha chains) found in anti-VI-immunoprecipitable material from hepatoma cell medium. Several lines of evidence suggest that the alpha E subunit, alone or incorporated into fibrinogen, is more stable than the common alpha chain, a feature of potential clinical importance.
Chromophore-assisted laser inactivation of proteins is mediated by the photogeneration of free radicals.
Authors: Liao J C , Roider J , Jay D G
Published: May 2006

Chromophore-assisted laser inactivation (CALI) is a technique that selectively inactivates proteins of interest to elucidate their in vivo functions. This method has application to a wide array of biological questions and an understanding of its mechanism is required for its judicious application. We report here that CALI is not mediated by photoinduced thermal denaturation but by photogenerated free radicals. Thermal diffusion calculations suggest that the temperature changes resulting from CALI are too small to cause thermal denaturation, and Arrhenius plots of CALI are inconsistent with a photothermal mechanism. CALI shows an energy dose reciprocity above a threshold and can be inhibited by free-radical quenchers, thus demonstrating a photochemical mechanism of protein inactivation. The type of quenchers that are effective in inhibiting CALI indicates that the active species is a hydrogen abstractor which is not derived from molecular oxygen. We suggest that the active free-radical species is the hydroxyl radical and its very short lifetime explains the spatial specificity of CALI such that half-maximal damage is effected within 15 A from the dye moiety and no significant damage occurs at 34 A. The data are consistent with free-radical formation resulting from a sequential two-photon process.
Structure of the human annexin VI gene.
Authors: Smith P D , Davies A , Crumpton M J , Moss S E
Published: May 2006

We report the structure of the human annexin VI gene and compare the intron-exon organization with the known structures of the human annexin I and II genes. The gene is approximately 60 kbp long and contains 26 exons. Consistent with the published annexin VI cDNA sequence, the genomic sequence at the 3' end does not contain a canonical polyadenylation signal. The genomic sequence upstream of the transcription start site contains TATAA and CAAT motifs. The spatial organization of the exons does not reveal any obvious similarities between the two halves of the annexin VI gene. Comparison of the intron-exon boundary positions of the annexin VI gene with those of annexins I and II reveals that within the repeated domains the break points are perfectly conserved except for exon 8, which is one codon smaller in annexin II. The corresponding point in the second half of annexin VI is represented by two exons, exons 20 and 21. The latter exon is alternatively spliced, giving rise to two annexin VI isoforms that differ with respect to a 6-amino acid insertion at the start of repeat 7.
Double replacement: strategy for efficient introduction of subtle mutations into the murine Col1a-1 gene by homologous recombination in embryonic stem cells.
Authors: Wu H , Liu X , Jaenisch R
Published: May 2006

A subtle mutation that rendered type I collagen resistant to mammalian collagenase has been introduced into the murine Col1a-1 (recently redesignated Cola-1) gene by homologous recombination in embryonic stem (ES) cells. Initially, a "hit and run" procedure was used. Since two steps were required for introducing each mutation and more than one mutation was to be introduced in the same genomic region independently, we have developed a streamlined procedure that involves two sequential replacement-type homologous recombination events. In the first step, an internal deletion was introduced into the Col1a-1 locus along with the positive and negative selectable markers, neo and tk, to mark the region of interest. G418-resistant homologous recombinants were isolated and used in the second step in which the deleted Col1a-1 allele was replaced with a construct containing the desired mutation. Homologous recombinants containing the mutation were identified among the Tk- ES clones after selection with FIAU [1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)-5-iodouracil (called fialuridine)]. Approximately 10% of such clones contained the desired mutation. The double replacement procedure greatly reduces the time and amount of work required to introduce mutations independently into the same or closely linked regions. Once the homologous recombinants derived from the first step are established, the introduction of other mutations into the deleted region becomes a one-step procedure. For X number of introduced mutations, 2X selections are required with the "hit and run" approach, but only X + 1 are required with the double-replacement method. This innovative procedure could be very useful in studies of gene structure and function as well as gene expression and regulation.
T-cell antigen CD28 interacts with the lipid kinase phosphatidylinositol 3-kinase by a cytoplasmic Tyr(P)-Met-Xaa-Met motif.
Authors: Prasad K V , Cai Y C , Raab M , Duckworth B , Cantley L , Shoelson S E , Rudd C E
Published: May 2006

The T-cell antigen CD28 provides a costimulatory signal that is required for T-cell proliferation. T-cell receptor zeta/CD3 engagement without CD28 ligation leads to a state of nonresponsiveness/anergy, thereby implicating CD28 in the control of peripheral tolerance to foreign antigens or tumors. A key unresolved question has concerned the mechanism by which CD28 generates intracellular signals. Phosphatidylinositol 3-kinase (PI 3-kinase) is a lipid kinase with Src-homology 2 (SH2) domain(s) that binds to the platelet-derived growth factor receptor (PDGF-R), an interaction that is essential for signaling by growth factor. In this study, we demonstrate that CD28 binds to PI 3-kinase by means of a Y(P)MXM motif within its cytoplasmic tail. CD28-associated PI 3-kinase was detected by lipid kinase and HPLC analysis as well as by reconstitution experiments with baculoviral-expressed p85 subunit of PI 3-kinase. CD28 bound directly to the p85 subunit without the need for the associated p110 subunit. Site-directed mutagenesis and peptide competition analysis using Y(P)-MXM-containing peptides showed that PI 3-kinase bound to a Y(P)MXM motif within the CD28 cytoplasmic tail (residues 191-194). Mutation of the Y191 within the motif resulted in a complete loss of binding, while mutation of M194 caused partial loss of binding. Binding analysis showed that the CD28 Y(P)-MXM motif bound to the p85 C- and N-terminal SH2 domains with an affinity comparable to that observed for PDGF-R and insulin receptor substrate 1. In terms of signaling, CD28 ligation induced a dramatic increase in the recruitment and association of PI 3-kinase with the receptor. CD28 is likely to use PI 3-kinase as the second signal leading to T-cell proliferation, an event with implications for anergy and peripheral T-cell tolerance.
Mechanosensitive Ca2+ transients in endothelial cells from human umbilical vein.
Authors: Oike M , Droogmans G , Nilius B
Published: May 2006

We have investigated the changes in intracellular calcium concentration ([Ca2+]i) in human endothelial cells induced by mechanical stretch due to osmotic cell swelling. Hypotonic solutions also activate a Cl- conductance that has been described elsewhere and mainly serves to clamp the membrane potential at negative values to provide a driving force for Ca2+ influx. The increase in [Ca2+]i caused by hypotonic solutions is due to release from inositol-1,4,5-trisphosphate-sensitive Ca2+ pools and a subsequent Ca2+ influx, apparently activated by store depletion. These changes in [Ca2+]i are completely abolished if the phospholipase A2 (PLA2) activity is inhibited by either 4-bromophenacyl bromide or cyclosporin A. Arachidonic acid, applied either extracellularly or intracellularly via the patch pipette, mimics the mechanosensitive response even in cells with blocked PLA2. Metabolites of the lipo- and cyclooxygenase pathways can be excluded. Phospholipase C activation and the protein kinase A pathway are not involved in this mechanical response. Although no specific pharmacological tools for probing the role of PLA2 are available, our evidence suggests that mechanosensitivity in endothelial cells may be modulated by arachidonic acid.
Ankyrin binds to two distinct cytoplasmic domains of Na,K-ATPase alpha subunit.
Authors: Devarajan P , Scaramuzzino D A , Morrow J S
Published: May 2006

Ankyrin has emerged as a ubiquitous protein linking integral membrane transport proteins such as Na,K-ATPase to an underlying spectrin cytoskeleton. This interaction is mediated by the alpha subunit of Na,K-ATPase; however, the nature of the ankyrin binding site in Na,K-ATPase is unknown. As a step to determine the mechanism of this interaction, the ankyrin binding region of human erythrocyte spectrin and each of five putative cytoplasmic domains of the Na,K-ATPase alpha subunit have been prepared as recombinant fusion proteins in bacteria and analyzed for their interaction with erythrocyte and kidney ankyrin (Ank1 and Ank3, respectively) in vitro. Spectrin binds both Ank1 and Ank3 avidly, as expected. Two of the Na,K-ATPase domains, immobilized on a bioaffinity column, also interact specifically with both of these ankyrins. These ATPase domains are encoded by codons 140-290 (domain II) and 345-784 (domain III), with domain II displaying the greatest apparent affinity. Sequences in domain II are highly conserved between species and isoforms of Na,K-ATPase and are homologous to a cytoplasmic domain in H,K-ATPase and to a limited region of sequence in Ca-ATPase. Conversely, domain II shares no significant homology with other ankyrin binding proteins such as band 3 and Na(+)-channel proteins. These results identify a clear function for a conserved but previously not understood region of the alpha subunit of Na,K-ATPase and suggest that the interaction of ankyrin with membrane transport proteins may involve complex tertiary structural determinants not easily deduced from the primary sequence.
Gene therapy for brain tumors: regression of experimental gliomas by adenovirus-mediated gene transfer in vivo.
Authors: Chen S H , Shine H D , Goodman J C , Grossman R G , Woo S L
Published: May 2006

The therapeutic efficacy of adenovirus-mediated herpes simplex virus thymidine kinase (HSV-tk) gene transduction of rat C6 glioma cells followed by ganciclovir (GCV) administration was studied in tumors generated in the brains of nude mice. C6 glioma cells were efficiently transduced in vitro by a replicative-defective recombinant adenovirus carrying the HSV-tk gene (ADV/RSV-tk) that rendered them sensitive to GCV in a dose-dependent manner. Tumors were generated by stereotaxic intracerebral injection of 1 x 10(4) C6 cells in nude mice. After 8 days of tumor growth, 3 x 10(8) ADV/RSV-tk viral particles were injected into the tumors and the mice subsequently were treated with GCV for 6 days. Tumor size in untreated and treated animals was compared 20 days after tumor implantation. The mean cross-sectional area of the tumors in the treated animals was 23-fold smaller than in control animals and the tumor volume was reduced by > 500-fold. These results demonstrate that the recombinant adenoviral vector can function as an efficient gene delivery vehicle for the treatment of gliomas by in vivo gene therapy.