bluuebunny/crossref_metadata_2025 huggingface dataset:
Characteristic enrichment of DNA repeats in different genomes
Authors: Cox Randal , Mirkin Sergei M.
Published: July 2002

Using computer programs developed for this purpose, we
searched for various repeated sequences including inverted, direct
tandem, and homopurine–homopyrimidine mirror repeats in various
prokaryotes, eukaryotes, and an archaebacterium. Comparison of observed
frequencies with expectations revealed that in bacterial genomes and
organelles the frequency of different repeats is either random or
enriched for inverted and/or direct tandem repeats. By contrast, in
all eukaryotic genomes studied, we observed an overrepresentation of
all repeats, especially homopurine–homopyrimidine mirror repeats.
Analysis of the genomic distribution of all abundant repeats showed
that they are virtually excluded from coding sequences. Unexpectedly,
the frequencies of abundant repeats normalized for their expectations
were almost perfect exponential functions of their size, and for a
given repeat this function was indistinguishable between different
genomes.
Transactivation by CIITA, the type II bare lymphocyte syndrome-associated factor, requires participation of multiple regions of the TATA box binding protein
Authors: Mahanta Sanjeev K. , Scholl Thomas , Yang Fu-Chia , Strominger Jack L.
Published: July 2002

CIITA is a positive regulator of class II major histocompatibility complex gene transcription that has been found to be defective in one of the five complementation groups of class II major histocompatibility complex-negative cell lines. Its N-terminal region is capable of activating transcription from a reporter gene when fused to a DNA binding domain. We have investigated the mechanism of transactivation mediated by the CIITA activation domain by studying its role in the process of transcription initiation and elongation. Specifically the altered specificity TBP (TATA box binding protein) assay has been used to analyze the response of the CIITA activation domain to mutations in TBP known to disrupt its interaction with its associated general factors. Transactivation by CIITA was extremely sensitive to a mutation in TBP that in yeast is known to abolish VP16-mediated transcription but leaves basal transcription unaffected. A TBP mutant defective in interaction with TBP-associated factor TAFII250 also failed to mediate transactivation through the CIITA activation domain. Certain interactions between TBP and general factors that are specifically required for acidic activation domains were also required for CIITA-mediated transactivation to reach its full potential. Finally, like VP16, CIITA was able to stimulate elongation of transcription. Overall the mechanism of transactivation by the human B-cell-specific CIITA is very similar to that mediated by the herpes virus transactivator VP16 in the ways that have been tested.
Mutagenicity in
Escherichia coli
of the major DNA adduct derived from the endogenous mutagen malondialdehyde
Authors: Fink Stephen P. , Reddy G. Ramachandra , Marnett Lawrence J.
Published: July 2002

The spectrum of mutations induced by the naturally occurring DNA adduct pyrimido[1,2-α]purin-10(3
H
)-one (M
1
G) was determined by site-specific approaches using M13 vectors replicated in
Escherichia coli
. M
1
G was placed at position 6256 in the (−)-strand of M13MB102 by ligating the oligodeoxynucleotide 5′-GGT(M
1
G)TCCG-3′ into a gapped-duplex derivative of the vector. Unmodified and M
1
G-modified genomes containing either a cytosine or thymine at position 6256 of the (+)-strand were transformed into repair-proficient and repair-deficient
E. coli
strains, and base pair substitutions were quantitated by hybridization analysis. Modified genomes containing a cytosine opposite M
1
G resulted in roughly equal numbers of M
1
G→A and M
1
G→T mutations with few M
1
G→C mutations. The total mutation frequency was ≈1%, which represents a 500-fold increase in mutations compared with unmodified M13MB102. Transformation of modified genomes containing a thymine opposite M
1
G allowed an estimate to be made of the ability of M
1
G to block replication. The (−)-strand was replicated >80% of the time in the unadducted genome but only 20% of the time when M
1
G was present. Correction of the mutation frequency for the strand bias of replication indicated that the actual frequency of mutations induced by M
1
G was 18%. Experiments using
E. coli
with different genetic backgrounds indicated that the SOS response enhances the mutagenicity of M
1
G and that M
1
G is a substrate for repair by the nucleotide excision repair complex. These studies indicate that M
1
G, which is present endogenously in DNA of healthy human beings, is a strong block to replication and an efficient premutagenic lesion.
Identification of an intramolecular interaction between small regions in type V adenylyl cyclase that influences stimulation of enzyme activity by G
sα
Authors: Scholich Klaus , Wittpoth Claus , Barbier Ann J. , Mullenix Jason B. , Patel Tarun B.
Published: July 2002

Using the full-length and two engineered soluble forms (C1-C2 and Cla-C2) of type V adenylyl cyclase (ACV), we have investigated the role of an intramolecular interaction in ACV that modulates the ability of the α subunit of the stimulatory GTP-binding protein of AC (G
sα
) to stimulate enzyme activity. Concentration–response curves with G
sα
suggested the presence of high and low affinity sites on ACV, which interact with the G protein. Activation of enzyme by G
sα
interaction at these two sites was most apparent in the C1a-C2 form of ACV, which lacks the C1b region (K
572
–F
683
). Yeast two-hybrid data demonstrated that the C1b region interacted with the C2 region and its 64-aa subdomain, C2I. Using peptides corresponding to the C2I region of ACV, we investigated the role of the C1b/C2I interaction on G
sα
-mediated stimulation of C1-C2 and full-length ACV. Our data demonstrate that a 10-aa peptide corresponding to L
1042
–T
1051
alters the profile of the activation curves of full-length and C1-C2 forms of ACV by different G
sα
concentrations to mimic the activation profile observed with C1a-C2 ACV. The various peptides used in our studies did not alter forskolin-mediated stimulation of full-length and C1-C2 forms of ACV. We conclude that the C1b region of ACV interacts with the 10-aa region (L
1042
–T
1051
) in the C2 domain of the enzyme to modulate G
sα
-elicited stimulation of activity.
Phosphorylation of the immunosuppressant FK506-binding protein FKBP52 by casein kinase II: Regulation of HSP90-binding activity of FKBP52
Authors: Miyata Yoshihiko , Chambraud Béatrice , Radanyi Christine , Leclerc Jocelyne , Lebeau Marie-Claire , Renoir Jack-Michel , Shirai Ryuichi , Catelli Maria-Grazia , Yahara Ichiro , Baulieu Etienne-Emile
Published: July 2002

FKBP52 (HSP56, p59, HBI) is the 59-kDa immunosuppressant FK506-binding protein and has peptidyl prolyl isomerase as well as a chaperone-like activity
in vitro
. FKBP52 associates with the heat shock protein HSP90 and is included in the steroid hormone receptor complexes
in vivo
. FKBP52 possesses a well conserved phosphorylation site for casein kinase II (CK2) that was previously shown to be associated with HSP90. Here we examined whether FKBP52 is phosphorylated by CK2 both
in vivo
and
in vitro
. Recombinant rabbit FKBP52 was phosphorylated by purified CK2. We expressed and purified deletion mutants of FKBP52 to determine the site(s) phosphorylated by CK2. Thr-143 in the hinge I region was identified as the major phosphorylation site for CK2. A synthetic peptide corresponding to this region was phosphorylated by CK2, and the peptide competitively inhibited the phosphorylation of other substrates by CK2. The [
32
P]phosphate labeling of FKBP52-expressing cells revealed that the same site is also phosphorylated
in vivo
. FK506 binding to FKBP52 did not affect the phosphorylation by CK2 and, conversely, the FK506-binding activity of FKBP52 was not affected by the phosphorylation. Most importantly, CK2-phosphorylated FKBP52 did not bind to HSP90. These results indicate that CK2 phosphorylates FKBP52 both
in vitro
and
in vivo
and thus may regulate the protein composition of chaperone-containing complexes such as those of steroid receptors and certain protein kinases.
Slow and fast dietary proteins differently modulate postprandial protein accretion
Authors: Boirie Yves , Dangin Martial , Gachon Pierre , Vasson Marie-Paule , Maubois Jean-Louis , Beaufrère Bernard
Published: July 2002

The speed of absorption of dietary amino acids by the gut varies according to the type of ingested dietary protein. This could affect postprandial protein synthesis, breakdown, and deposition. To test this hypothesis, two intrinsically
13
C-leucine-labeled milk proteins, casein (CAS) and whey protein (WP), of different physicochemical properties were ingested as one single meal by healthy adults. Postprandial whole body leucine kinetics were assessed by using a dual tracer methodology. WP induced a dramatic but short increase of plasma amino acids. CAS induced a prolonged plateau of moderate hyperaminoacidemia, probably because of a slow gastric emptying. Whole body protein breakdown was inhibited by 34% after CAS ingestion but not after WP ingestion. Postprandial protein synthesis was stimulated by 68% with the WP meal and to a lesser extent (+31%) with the CAS meal. Postprandial whole body leucine oxidation over 7 h was lower with CAS (272 ± 91 μmol⋅kg
−1
) than with WP (373 ± 56 μmol⋅kg
−1
). Leucine intake was identical in both meals (380 μmol⋅kg
−1
). Therefore, net leucine balance over the 7 h after the meal was more positive with CAS than with WP (
P
< 0.05, WP vs. CAS). In conclusion, the speed of protein digestion and amino acid absorption from the gut has a major effect on whole body protein anabolism after one single meal. By analogy with carbohydrate metabolism, slow and fast proteins modulate the postprandial metabolic response, a concept to be applied to wasting situations.
DNA double-strand-break sensitivity, DNA replication, and cell cycle arrest phenotypes of Ku-deficient
Saccharomyces cerevisiae
Authors: Barnes Georjana , Rio Donald
Published: July 2002

In mammalian cells, the Ku heterodimer is involved in DNA double-strand-break recognition and repair. We have established in yeast a connection between Ku activity and DNA double-strand-break damage repair, and a connection between Ku activity and commitment to DNA replication. We generated double-stranded DNA breaks in yeast cells
in vivo
by expressing a restriction endonuclease and have shown that yeast mutants lacking Ku p70 activity died while isogenic wild-type cells did not. Moreover, we have discovered that DNA damage occurs spontaneously during normal yeast mitotic growth, and that Ku functions in repair of this damage. We also observed that mitotically growing Ku p70 mutants have an anomalously high DNA content, suggesting a role for Ku in regulation of DNA synthesis. Finally, we present evidence that Ku p70 function is conserved between yeast,
Drosophila
, and humans.
Schizophrenia is associated with elevated amphetamine-induced synaptic dopamine concentrations: Evidence from a novel positron emission tomography method
Authors: Breier A. , Su T.-P. , Saunders R. , Carson R. E. , Kolachana B. S. , de Bartolomeis A. , Weinberger D. R. , Weisenfeld N. , Malhotra A. K. , Eckelman W. C. , Pickar D.
Published: July 2002

A major line of evidence that supports the hypothesis of dopamine overactivity in schizophrenia is the psychomimetic potential of agents such as amphetamine that stimulate dopamine outflow. A novel brain imaging method provides an indirect measure of
in vivo
synaptic dopamine concentration by quantifying the change in dopamine receptor radiotracer binding produced by agents that alter dopamine release but do not themselves bind to dopamine receptors. The purpose of this investigation is (
i
) to determine the sensitivity (i.e., amount of dopamine reflected in radiotracer binding changes) of this method by examining the relationship between amphetamine-induced changes in simultaneously derived striatal extracellular dopamine levels with
in vivo
microdialysis and striatal binding levels with the dopamine D
2
/D
3
positron-emission tomography radioligand [
11
C]raclopride in nonhuman primates, and (
ii
) to test the hypothesis of elevated amphetamine-induced synaptic dopamine levels in schizophrenia. In the nonhuman primate study (
n
= 4), doubling the amphetamine dose produced a doubling in [
11
C]raclopride specific binding reductions. In addition, the ratio of percent mean dopamine increase to percent mean striatal binding reduction for amphetamine (0.2 mg/kg) was 44:1, demonstrating that relatively small binding changes reflect large changes in dopamine outflow. In the clinical study, patients with schizophrenia (
n
= 11) compared with healthy volunteers (
n
= 12) had significantly greater amphetamine-related reductions in [
11
C]raclopride specific binding (mean ± SEM): −22.3% (±2.7) vs. −15.5% (±1.8),
P
= 0.04, respectively. Inferences from the preclinical study suggest that the patients’ elevation in synaptic dopamine concentrations was substantially greater than controls. These data provide direct evidence for the hypothesis of elevated amphetamine-induced synaptic dopamine concentrations in schizophrenia.
On the difference in ionization properties between planar interfaces and linear polyelectrolytes
Authors: Borkovec Michal , Daicic John , Koper Ger J. M.
Published: July 2002

Ionizable planar interfaces and linear polyelectrolytes show
markedly different proton-binding behavior. Planar interfaces protonate
in a single broad step, whereas polyelectrolytes mostly undergo a
two-step protonation. Such contrasting behavior is explained using a
discrete-charge Ising model. This model is based on an approximation of
the ionizable groups by point charges that are treated within a
linearized Poisson–Boltzmann approximation. The underlying reason as
to why planar interfaces exhibit mean-field-like behavior, whereas
linear polyelectrolytes usually do not, is related to the range of the
site–site interaction potential. For a planar interface, this
interaction potential is much more long ranged if compared with that of
the cylindrical geometry as appropriate to a linear polyelectrolyte.
The model results are in semi-quantitative agreement with experimental
data for fatty-acid monolayers, water-oxide interfaces, and various
linear polyelectrolytes.
Normal viability and altered pharmacokinetics in mice lacking mdr1-type (drug-transporting) P-glycoproteins
Authors: Schinkel Alfred H. , Mayer Ulrich , Wagenaar Els , Mol Carla A. A. M. , van Deemter Liesbeth , Smit Jaap J. M. , van der Valk Martin A. , Voordouw Arie C. , Spits Hergen , van Tellingen Olaf , Zijlmans J. Mark J. M. , Fibbe Willem E. , Borst Piet
Published: July 2002

The mdr1-type P-glycoproteins (P-gps) confer multidrug resistance to cancer cells by active extrusion of a wide range of drugs from the cell. To study their physiological roles, we have generated mice genetically deficient in themdr1bgene [mdr1b(−/−) mice] and in both themdr1aandmdr1bgenes [mdr1a/1b(−/−) mice]. In spite of the host of functions speculatively attributed to the mdr1-type P-gps, we found no physiological abnormalities in either strain. Viability, fertility, and a range of histological, hematological, serum–chemical, and immunological parameters were not abnormal inmdr1a/1b(−/−) mice. The high level of mdr1b P-gp normally present in the pregnant uterus did not protect fetuses from a drug (digoxin) in the bloodstream of the mother, although the protein did reduce drug accumulation in the adrenal gland and ovaries. Pharmacologically,mdr1a/1b(−/−) mice behaved similarly to the previously analyzedmdr1a(−/−) mice, displaying, for instance, increased brain penetration and reduced elimination of digoxin. However, both mdr1a and mdr1b P-gps contributed to the extrusion of rhodamine from hematopoietic progenitor cells, suggesting a potential role for the endogenous mdr1-type P-gps in protection of bone marrow against cytotoxic anticancer drugs. This, and the normal viability ofmdr1a/1b(−/−) mice, has implications for the use of P-gp-blocking agents in cancer and other chemotherapy.mdr1a/1b(−/−) mice should provide a useful model system to further test the pharmacological roles of the drug-transporting P-gps and to analyze the specificity and effectivity of P-gp-blocking drugs.
Carcinogens induce reversion of the mouse pink-eyed unstable mutation
Authors: Schiestl Robert H. , Aubrecht Jiri , Khogali Fathia , Carls Nicholas
Published: July 2002

Deletions and other genome rearrangements are associated with carcinogenesis and inheritable diseases. The pink-eyed unstable (
p
un
) mutation in the mouse is caused by duplication of a 70-kb internal fragment of the
p
gene. Spontaneous reversion events in homozygous
p
un
/
p
un
mice occur through deletion of a duplicated sequence. Reversion events in premelanocytes in the mouse embryo detected as black spots on the gray fur of the offspring were inducible by the carcinogen x-rays, ethyl methanesulfonate, methyl methanesulfonate, ethyl nitrosourea, benzo[
a
]pyrene, trichloroethylene, benzene, and sodium arsenate. The latter three carcinogens are not detectable with several
in vitro
or
in vivo
mutagenesis assays. We studied the molecular mechanism of the carcinogen-induced reversion events by cDNA analysis using reverse transcriptase–PCR method and identified the induced reversion events as deletions. DNA deletion assays may be sensitive indicators for carcinogen exposure.
Signal-dependent translation of a regulatory protein, Bcl-3, in activated human platelets
Authors: Weyrich Andrew S. , Dixon Dan A. , Pabla Ravinder , Elstad Mark R. , McIntyre Thomas M. , Prescott Stephen M. , Zimmerman Guy A.
Published: July 2002

Circulating human platelets lack nuclei, cannot synthesize mRNA, and are considered incapable of regulated protein synthesis. We found that thrombin-activated, but not resting, platelets synthesize Bcl-3, a member of the IκB-α family of regulatory proteins. The time- and concentration-dependent generation of Bcl-3 in platelets signaled by thrombin was blocked by translational inhibitors, by rapamycin, and by inhibitors of phosphatidylinositol-3-kinase, indicating that it occurs via a specialized translational control pathway that involves phosphorylation of the inhibitory protein 4E-BP1. After its synthesis in activated platelets Bcl-3 binds to the SH3 domain of Fyn (p59

fyn

), a Src-related tyrosine kinase. This, along with its expression in anucleate cells, suggests that Bcl-3 has previously unrecognized functions aside from modulation of transcription. We also demonstrate that platelets synthesize and secrete numerous proteins besides Bcl-3 after they adhere to fibrinogen, which mediates adhesion and outside–in signaling of these cells by engagement of αIIb/β3 integrin. Taken together, these data demonstrate that regulated synthesis of proteins is a signal-dependent activation response of human platelets.
Angiostatin gene transfer: Inhibition of tumor growth
in vivo
by blockage of endothelial cell proliferation associated with a mitosis arrest
Authors: Griscelli Frank , Li Hong , Bennaceur-Griscelli Annelise , Soria Jeannette , Opolon Paule , Soria Claudine , Perricaudet Michel , Yeh Patrice , Lu He
Published: July 2002

The antitumoral effects that follow the local delivery of the N-terminal fragment of human plasminogen (angiostatin K3) have been studied in two xenograft murine models. Angiostatin delivery was achieved by a defective adenovirus expressing a secretable angiostatin K3 molecule from the cytomegalovirus promoter (AdK3). In
in vitro
studies, AdK3 selectively inhibited endothelial cell proliferation and disrupted the G
2
/M transition induced by M-phase-promoting factors. AdK3-infected endothelial cells showed a marked mitosis arrest that correlated with the down-regulation of the M-phase phosphoproteins. A single intratumoral injection of AdK3 into preestablished rat C6 glioma or human MDA-MB-231 breast carcinoma grown in athymic mice was followed by a significant arrest of tumor growth, which was associated with a suppression of neovascularization within and at the vicinity of the tumors. AdK3 therapy also induced a 10-fold increase in apoptotic tumor cells as compared with a control adenovirus. Furthermore, we showed that systemic injection of AdK3 delayed C6 tumor establishment and growth, confirming that angiostatin can function in a paracrin manner. Our data support the concept that targeted antiangiogenesis, using adenovirus-mediated gene transfer, represents a promising alternative strategy for delivering antiangiogenic factors as their bolus injections present unsolved pharmacological problems.
Functional transitions in myosin: Formation of a critical salt-bridge and transmission of effect to the sensitive tryptophan
Authors: Onishi Hirofumi , Kojima Shin-ichiro , Katoh Kazuo , Fujiwara Keigi , Martinez Hugo M. , Morales Manuel F.
Published: July 2002

For analyzing the mechanism of energy transduction in the “motor” protein, myosin, it is opportune both to model the structural change in the hydrolytic transition, ATP (myosin-bound) + H
2
O → ADP⋅P
i
(myosin-bound) and to check the plausibility of the model by appropriate site-directed mutations in the functional system. Here, we made a series of mutations to investigate the role of the salt-bridge between Glu-470 and Arg-247 (of chicken smooth muscle myosin) that has been inferred from crystallography to be a central feature of the transition [Fisher, A. J., Smith, C. A., Thoden, J. B., Smith, R., Sutoh, K., Holden, H. M., & Rayment, I. (1995)
Biochemistry
34, 8960–8972]. Our results suggest that whether in the normal, or in the inverted, direction an intact salt-bridge is necessary for ATP hydrolysis, but when the salt-bridge is in the inverted direction it does not support actin activation. Normally, fluorescence changes result from adding nucleotides to myosin; these signals are reported by Trp-512 (of chicken smooth muscle myosin). Our results also suggest that structural impairments in the 470–247 region interfere with the transmission of these signals to the responsive Trp.
Interaction of SP100 with HP1 proteins: A link between the promyelocytic leukemia-associated nuclear bodies and the chromatin compartment
Authors: Seeler Jacob-S. , Marchio Agnès , Sitterlin Delphine , Transy Catherine , Dejean Anne
Published: July 2002

The PML/SP100 nuclear bodies (NBs) were first described as discrete subnuclear structures containing the SP100 protein. Subsequently, they were shown to contain the PML protein which is part of the oncogenic PML-RARα hybrid produced by the t(15;17) chromosomal translocation characteristic of acute promyelocytic leukemia. Yet, the physiological role of these nuclear bodies remains unknown. Here, we show that SP100 binds to members of the heterochromatin protein 1 (HP1) families of non-histone chromosomal proteins. Further, we demonstrate that a naturally occurring splice variant of SP100, here called SP100-HMG, is a member of the high mobility group-1 (HMG-1) protein family and may thus possess DNA-binding potential. Both HP1 and SP100-HMG concentrate in the PML/SP100 NBs, and overexpression of SP100 leads to enhanced accumulation of endogenous HP1 in these structures. When bound to a promoter, SP100, SP100-HMG and HP1 behave as transcriptional repressors in transfected mammalian cells. These observations present molecular evidence for an association between the PML/SP100 NBs and the chromatin nuclear compartment. They support a model in which the NBs may play a role in certain aspects of chromatin dynamics.
Cannabidiol and (−)Δ
9
-tetrahydrocannabinol are neuroprotective antioxidants
Authors: Hampson A. J. , Grimaldi M. , Axelrod J. , Wink D.
Published: July 2002

The neuroprotective actions of cannabidiol and other cannabinoids were examined in rat cortical neuron cultures exposed to toxic levels of the excitatory neurotransmitter glutamate. Glutamate toxicity was reduced by both cannabidiol, a nonpsychoactive constituent of marijuana, and the psychotropic cannabinoid (−)Δ
9
-tetrahydrocannabinol (THC). Cannabinoids protected equally well against neurotoxicity mediated by N-methyl-
d
-aspartate receptors, 2-amino-3-(4-butyl-3-hydroxyisoxazol-5-yl)propionic acid receptors, or kainate receptors. N-methyl-
d
-aspartate receptor-induced toxicity has been shown to be calcium dependent; this study demonstrates that 2-amino-3-(4-butyl-3-hydroxyisoxazol-5-yl)propionic acid/kainate receptor-type neurotoxicity is also calcium-dependent, partly mediated by voltage sensitive calcium channels. The neuroprotection observed with cannabidiol and THC was unaffected by cannabinoid receptor antagonist, indicating it to be cannabinoid receptor independent. Previous studies have shown that glutamate toxicity may be prevented by antioxidants. Cannabidiol, THC and several synthetic cannabinoids all were demonstrated to be antioxidants by cyclic voltametry. Cannabidiol and THC also were shown to prevent hydroperoxide-induced oxidative damage as well as or better than other antioxidants in a chemical (Fenton reaction) system and neuronal cultures. Cannabidiol was more protective against glutamate neurotoxicity than either ascorbate or α-tocopherol, indicating it to be a potent antioxidant. These data also suggest that the naturally occurring, nonpsychotropic cannabinoid, cannabidiol, may be a potentially useful therapeutic agent for the treatment of oxidative neurological disorders such as cerebral ischemia.
The solution structure of the DNA-binding domain of Skn-1
Authors: Lo Mei-Chu , Ha Sha , Pelczer István , Pal Santona , Walker Suzanne
Published: July 2002

Skn-1 is a maternally expressed transcription factor that specifies the fate of certain blastomeres early in the development of
Caenorhabditis elegans.
It has been reported that the DNA-binding domain is a molten globule and that the structure cannot be defined because there are no long-range nuclear Overhauser effects (NOEs). Working with short Skn domain fragments and using
13
C-labeled proteins, we have been able to identify 28 long-range NOEs that establish a tertiary fold for the Skn domain. The internal region of the Skn domain consists of three stable helices and one conformationally labile helix organized into a nascent helix–turn–helix–turn–helix–turn–helix motif. The N and C termini of the Skn domain are unstructured and emerge from the same end of the folded domain. This structure is consistent with biochemical data on binding of the Skn domain to DNA, which shows that the N and C termini bind in the adjacent minor and major grooves from the same face of the DNA helix. The NMR solution structure of the Skn domain should be useful for developing a complete understanding of the DNA recognition event, including any conformational changes that take place upon binding.
Brain-derived neurotrophic factor modulates hippocampal synaptic transmission by increasing
N
-methyl-
d
-aspartic acid receptor activity
Authors: Levine Eric S. , Crozier Robert A. , Black Ira B. , Plummer Mark R.
Published: July 2002

Neurotrophins (NTs) have recently been found to regulate synaptic transmission in the hippocampus. Whole-cell and single-channel recordings from cultured hippocampal neurons revealed a mechanism responsible for enhanced synaptic strength. Specifically, brain-derived neurotrophic factor augmented glutamate-evoked, but not acetylcholine-evoked, currents 3-fold and increased
N
-methyl-
d
-aspartic acid (NMDA) receptor open probability. Activation of trkB NT receptors was critical, as glutamate currents were not affected by nerve growth factor or NT-3, and increased open probability was prevented by the tyrosine kinase inhibitor K-252a. In addition, the NMDA receptor antagonist MK-801 blocked brain-derived neurotrophic factor enhancement of synaptic transmission, further suggesting that NTs modulate synaptic efficacy via changes in NMDA receptor function.
Overexpression of the large subunit of the protein Ku suppresses metallothionein-I induction by heavy metals
Authors: Ghoshal Kalpana , Li Zhiling , Jacob Samson T.
Published: July 2002

Metallothioneins (MT) are involved in the scavenging of the toxic heavy metals and protection of cells from reactive oxygen intermediates. To investigate the potential role of the protein Ku in the expression of MT, we measured the level of MT-I mRNA in the parental rat fibroblast cell line (Rat 1) and the cell lines that stably and constitutively overexpress the small subunit, the large subunit, and the heterodimer of Ku. Treatment with CdS0
4
or ZnS0
4
elevated the MT-I mRNA level 20- to 30-fold in the parental cells and the cells (Ku-70) that overproduce the small subunit or those (Ku-7080) overexpressing the heterodimer. By contrast, the cells (Ku-80) overexpressing the large subunit of Ku failed to induce MT-I.
In vitro
transcription assay showed that the MT-I promoter activity was suppressed selectively in the nuclear extracts from Ku-80 cells. The specificity of the repressor function was shown by the induction of hsp 70, another Cd-inducible gene, in Ku-80 cells. Addition of the nuclear extract from Ku-80 cells at the start of the transcription reaction abolished the MT-l promoter activity in the Rat 1 cell extract. The transcript once formed in Rat 1 nuclear extract was not degraded by further incubation with the extract from Ku-80 cells. The repressor was sensitive to heat. The DNA-binding activities of at least four transcription factors that control the MT-I promoter activity were not affected in Ku-80 cells. These observations have set the stage for further exploration of the mechanisms by which the Ku subunit mediates suppression of MT induction.
Zinc-dependent dimers observed in crystals of human endostatin
Authors: Ding Yuan-Hua , Javaherian Kashi , Lo Kin-Ming , Chopra Rajiv , Boehm Thomas , Lanciotti Julia , Harris B. A. , Li Yue , Shapiro Robert , Hohenester Erhard , Timpl Rupert , Folkman Judah , Wiley Don C.
Published: July 2002

The crystal structure of human endostatin reveals a zinc-binding
site. Atomic absorption spectroscopy indicates that zinc is a
constituent of both human and murine endostatin in solution. The human
endostatin zinc site is formed by three histidines at the N terminus,
residues 1, 3, and, 11, and an aspartic acid at residue 76. The
N-terminal loop ordered around the zinc makes a dimeric contact in
human endostatin crystals. The location of the zinc site at the amino
terminus, immediately adjacent to the precursor cleavage site, suggests
the possibility that the zinc may be involved in activation of the
antiangiogenic activity following cleavage from the inactive collagen
XVIII precursor or in the cleavage process itself.
The natural polyamine spermine functions directly as a free radical scavenger
Authors: Ha Hyo Chol , Sirisoma Nilantha S. , Kuppusamy Periannan , Zweier Jay L. , Woster Patrick M. , Casero Robert A.
Published: July 2002

The polyamines are small organic cations that are absolutely required for eukaryotic cell growth. Although their growth requirements are well established, the molecular functions of the polyamines are ill-defined. Oxidative damage to DNA by reactive oxygen species is a continual problem that cells must guard against to survive. The polyamine spermine, which is normally found in millimolar concentrations in the nucleus, is shown here to function directly as a free radical scavenger, and adducts formed as a result of this function are identified. These data suggest that spermine is a major natural intracellular compound capable of protecting DNA from free radical attack.
Abscisic acid-induced stomatal closure mediated by cyclic ADP-ribose
Authors: Leckie Calum P. , McAinsh Martin R. , Allen Gethyn J. , Sanders Dale , Hetherington Alistair M.
Published: July 2002

Abscisic acid (ABA) is a plant hormone involved in the response of plants to reduced water availability. Reduction of guard cell turgor by ABA diminishes the aperture of the stomatal pore and thereby contributes to the ability of the plant to conserve water during periods of drought. Previous work has demonstrated that cytosolic Ca
2+
is involved in the signal transduction pathway that mediates the reduction in guard cell turgor elicited by ABA. Here we report that ABA uses a Ca
2+
-mobilization pathway that involves cyclic adenosine 5′-diphosphoribose (cADPR). Microinjection of cADPR into guard cells caused reductions in turgor that were preceded by increases in the concentration of free Ca
2+
in the cytosol. Patch clamp measurements of isolated guard cell vacuoles revealed the presence of a cADPR-elicited Ca
2+
-selective current that was inhibited at cytosolic Ca
2+
≥ 600 nM. Furthermore, microinjection of the cADPR antagonist 8-NH
2
-cADPR caused a reduction in the rate of turgor loss in response to ABA in 54% of cells tested, and nicotinamide, an antagonist of cADPR production, elicited a dose-dependent block of ABA-induced stomatal closure. Our data provide definitive evidence for a physiological role for cADPR and illustrate one mechanism of stimulus-specific Ca
2+
mobilization in higher plants. Taken together with other recent data [Wu, Y., Kuzma, J., Marechal, E., Graeff, R., Lee, H. C., Foster, R. & Chua, N.-H. (1997)
Science
278, 2126–2130], these results establish cADPR as a key player in ABA signal transduction pathways in plants.
HLA class I-specific inhibitory receptors in human T lymphocytes: Interleukin 15-induced expression of CD94/NKG2A in superantigen- or alloantigen-activated CD8+ T cells
Authors: Mingari Maria Cristina , Ponte Marco , Bertone Stefania , Schiavetti Francesca , Vitale Chiara , Bellomo Rosa , Moretta Alessandro , Moretta Lorenzo
Published: July 2002

A fraction of human T lymphocytes, predominantly CD8+, express receptors for HLA class I molecules typical of natural killer cells (natural killer receptors or NKRs) that inhibit T cell receptor-mediated functions. Herein, we analyzed possible mechanism(s) leading to the expression of NKRs by T cells responding to superantigens or allogeneic cellsin vitro. We show that, in the presence of interleukin 15 (IL-15), T cells (depleted of NKR+cells) responding to toxic shock syndrome toxin 1de novoexpress CD94, a molecule that is part of a heterodimeric NKR with a broad specificity for different HLA class I alleles. Maximal CD94 expression occurred when IL-15 was added shortly after the cells were placed into culture, and CD94 expression started 4–6 days after addition of IL-15. Although both CD4+and CD8+cells expressed CD94, the simultaneous expression of NKG2A (i.e., the other component of the CD94/NKG2A inhibitory NKR) was confined to CD8+cells. Similar data were obtained in T cell populations activated in mixed lymphocyte cultures in the presence of IL-15. The expression of CD94/NKG2A led to an impairment of allo-specific cytolytic activity by mixed lymphocyte culture-derived T cell populations or clones. Remarkably, cytolysis could be restored by the addition of anti-CD94 mAb, i.e., by masking the inhibitory NKRs.
Imaging neuroscience: Principles or maps?
Authors: Friston Karl J.
Published: July 2002

This article reviews some recent trends in imaging neuroscience. A
distinction is made between making maps of functional responses in the
brain and discerning the rules or principles that underlie their
organization. After considering developments in the characterization of
brain imaging data, several examples are presented that highlight the
context-sensitive nature of neuronal responses that we measure. These
contexts can be endogenous and physiological, reflecting
the fact that each cortical area, or neuronal population, expresses its
dynamics in the context of interactions with other areas. Conversely,
these contexts can be experimental or psychological and can have a
profound effect on the regional effects elicited. In this review we
consider experimental designs and analytic strategies that go beyond
cognitive subtraction and speculate on how functional imaging can be
used to address both the details and principles underlying functional
integration and specialization in the brain.
Characterization of a calmodulin-binding transporter from the plasma membrane of barley aleurone
Authors: Schuurink Robert C. , Shartzer Stacey F. , Fath Angelika , Jones Russell L.
Published: July 2002

We have used
Arabidopsis
calmodulin (CaM) covalently coupled to horseradish peroxidase to screen a barley aleurone cDNA expression library for CaM binding proteins. The deduced amino acid sequence of one cDNA obtained by this screen was shown to be a unique protein of 702 amino acids with CaM and cyclic nucleotide binding domains at the carboxyl terminus and high similarity to olfactory and K
+
channels. This cDNA was designated HvCBT1 (
Hordeum vulgare
CaM binding transporter). Hydropathy plots of HvCBT1 showed the presence of six putative transmembrane domains, but sequence alignment indicated a pore domain that was unlike the consensus domains in K
+
and olfactory channels. Expression of a subclone of amino acids 482–702 in
Escherichia coli
generated a peptide that bound CaM. When a fusion protein of HvCBT1 and green fluorescent protein was expressed in barley aleurone protoplasts, fluorescence accumulated in the plasma membrane. Expression of HvCBT1 in the K
+
transport deficient
Saccharomyces cerevisiae
mutant CY162 showed no rescue of the mutant phenotype. However, growth of CY162 expressing HvCBT1 with its pore mutated to GYGD, the consensus sequence of K
+
channels, was compromised. We interpret these data as indicating that HvCBT1 acts to interfere with ion transport.
Hypotension and inflammatory cytokine gene expression triggered by factor Xa–nitric oxide signaling
Authors: Papapetropoulos Andreas , Piccardoni Paola , Cirino Giuseppe , Bucci Mariarosaria , Sorrentino Raffaella , Cicala Carla , Johnson Kirk , Zachariou Venetia , Sessa William C. , Altieri Dario C.
Published: July 2002

The signaling pathway initiated by factor Xa on vascular endothelial cells was investigated. Factor Xa stimulated a 5- to 10-fold increased release of nitric oxide (NO) in a dose-dependent reaction (0.1–2.5 μg/ml) unaffected by the thrombin inhibitor hirudin but abolished by active site inhibitors, tick anticoagulant peptide, or Glu-Gly-Arg-chloromethyl ketone. In contrast, the homologous clotting protease factor IXa or another endothelial cell ligand, fibrinogen, was ineffective. A factor Xa inter-epidermal growth factor synthetic peptide L83FTRKL88(G) blocking ligand binding to effector cell protease receptor-1 inhibited NO release by factor Xa in a dose-dependent manner, whereas a control scrambled peptide KFTGRLL was ineffective. Catalytically active factor Xa induced hypotension in rats and vasorelaxation in the isolated rat mesentery, which was blocked by the NO synthase inhibitorl-NG-nitroarginine methyl ester (l-NAME) but not byd-NAME. Factor Xa/NO signaling also produced a dose-dependent endothelial cell release of interleukin 6 (range 0.55–3.1 ng/ml) in a reaction inhibited byl-NAME and by the inter-epidermal growth factor peptide Leu83–Leu88but unaffected by hirudin. Maximal induction of interleukin 6 mRNA required a brief, 30-min stimulation with factor Xa, unaffected by subsequent addition of tissue factor pathway inhibitor. These data suggest that factor Xa-induced NO release modulates endothelial cell-dependent vasorelaxation and cytokine gene expression. This pathway requiring factor Xa binding to effector cell protease receptor-1 and a secondary step of ligand-dependent proteolysis may preserve an anti-thrombotic phenotype of endothelium but also trigger acute phase responses during activation of coagulationin vivo.
Regulation of a nuclear export signal by an adjacent inhibitory sequence: The effector domain of the influenza virus NS1 protein
Authors: Li Yongzhong , Yamakita Yoshihiko , Krug Robert M.
Published: July 2002

In the cell nucleus the NS1 protein of influenza A virus inhibits both pre-mRNA splicing and the nuclear export of mRNAs. Both the RNA-binding and effector domains of the protein are required for these nuclear functions. Here we demonstrate that the NS1 protein has a latent nuclear export signal (NES) that is located at the amino end of the effector domain. In uninfected, transfected cells the NS1 protein is localized in the nucleus because the NES is specifically inhibited by the adjacent amino acid sequence in the effector domain. Substitution of alanine residues for specific amino acids in the adjacent sequence abrogates its inhibitory activity, thereby unmasking the NES and causing the full-length NS1 protein to be localized to the cytoplasm. In contrast to uninfected cells, a substantial amount of the NS1 protein in influenza virus-infected cells is located in the cytoplasm. Consequently, the NES of these NS1 protein molecules is unmasked in infected cells, indicating that the NS1 protein most likely carries out functions in the cytoplasm as well as the nucleus. A dramatically different localization of the NS1 protein occurs in cells that are infected by a virus encoding an NS1 protein lacking the NES: the shortened NS1 protein molecules are almost totally in the nucleus. Because the NES of the full-length NS1 protein is unmasked in infected but not uninfected cells, it is likely that this unmasking results from a specific interaction of another virus-specific protein with the NS1 protein.
Mechanism of recruitment of DnaB helicase to the replication origin of the plasmid pSC101
Authors: Datta Hirock J. , Khatri Ghan Shyam , Bastia Deepak
Published: July 2002

Although many bacterial chromosomes require only one replication initiator protein, e.g., DnaA, most plasmid replicons depend on dual initiators: host-encoded DnaA and plasmid-encoded Rep initiator protein for replication initiation. Using the plasmid pSC101 as a model system, this work investigates the biological rationale for the requirement for dual initiators and shows that the plasmid-encoded RepA specifically interacts with the replicative helicase DnaB. Mutations in DnaB or RepA that disrupt RepA–DnaB interaction cause failure to load DnaB to the plasmid
ori in vitro
and to replicate the plasmid
in vivo
. Although, interaction of DnaA with DnaB could not substitute for RepA–DnaB interaction for helicase loading, DnaA along with integration host factor, DnaC, and RepA was essential for helicase loading. Therefore, DnaA is indirectly needed for helicase loading. Instead of a common surface of interaction with initiator proteins, interestingly, DnaB helicase appears to have at least a limited number of nonoverlapping surfaces, each of which interacts specifically with a different initiator protein.
Megacystis, mydriasis, and ion channel defect in mice lacking the α3 neuronal nicotinic acetylcholine receptor
Authors: Xu Wei , Gelber Shari , Orr-Urtreger Avi , Armstrong Dawna , Lewis Richard A. , Ou Ching-Nan , Patrick James , Role Lorna , De Biasi Mariella , Beaudet Arthur L.
Published: July 2002

The α3 subunit of the neuronal nicotinic acetylcholine receptor
is widely expressed in autonomic ganglia and in some parts of the
brain. The α3 subunit can form heteromultimeric ion channels with
other α subunits and with β2 and β4 subunits, but its function
in vivo
is poorly understood. We prepared a null
mutation for the α3 gene by deletion of exon 5 and found that
homozygous (−/−) mice lacked detectable mRNA on Northern
blotting. The −/− mice survive to birth but have impaired
growth and increased mortality before and after weaning. The
−/− mice have extreme bladder enlargement, dribbling
urination, bladder infection, urinary stones, and widely dilated ocular
pupils that do not contract in response to light. Detailed histological
studies of −/− mice revealed no significant abnormalities in
brain or peripheral tissues except urinary bladder, where inflammation
was prominent. Ganglion cells and axons were present in bladder and
bowel. Bladder strips from −/− mice failed to contract in
response to 0.1 mM nicotine, but did contract in response to electrical
field stimulation or carbamoylcholine. The number of
acetylcholine-activated single-channel currents was severely reduced in
the neurons of superior cervical ganglia in −/− mice with five
physiologically distinguishable nicotinic acetylcholine receptor
subtypes with different conductance and kinetic properties in wild-type
mice, all of which were reduced in −/− mice. The findings in
the α3-null mice suggest that this subunit is an essential component
of the nicotinic receptors mediating normal function of the autonomic
nervous system. The phenotype in −/− mice may be similar to
the rare human genetic disorder of megacystis–microcolon–intestinal
hypoperistalsis syndrome.
Origin of a new
Phytophthora
pathogen through interspecific hybridization
Authors: Brasier C. M. , Cooke D. E. L. , Duncan J. M.
Published: July 2002

Plant disease epidemics resulting from introductions of exotic fungal plant pathogens are a well known phenomenon. An associated risk—that accelerated pathogen evolution may be occurring as a consequence of genetic exchange between introduced, or introduced and resident, fungal pathogens—is largely unrecognized. This is, in part, because examples of natural, interspecific hybridization in fungi are very rare. Potential evolutionary developments range from the acquisition of new host specificities to emergence of entirely new pathogen taxa. We present evidence from cytological behavior, additive nucleotide bases in repetitive internal transcribed spacer regions of the rRNA-encoding DNA (rDNA), and amplified fragment length polymorphisms of total DNA that a new, aggressive
Phytophthora
pathogen of alder trees in Europe comprises a range of heteroploid-interspecific hybrids involving a
Phytophthora cambivora
-like species and an unknown taxon similar to
Phytophthora fragariae
. The hybrids’ marked developmental instabilities, unusual morphological variability, and evidence for recombination in their internal transcribed spacer profiles indicates that they are of recent origin and that their evolution is continuing. The likelihood of such evolutionary events may be increasing as world trade in plants intensifies. However, routine diagnostic procedures currently in use are insufficiently sensitive to allow their detection.
Paternal monoallelic expression of the paired immunoglobulin-like receptors PIR-A and PIR-B
Authors: Chen Ching-Cheng , Hurez Vincent , Brockenbrough J. Scott , Kubagawa Hiromi , Cooper Max D.
Published: July 2002

A diverse pattern of polymorphism is defined for the paired Ig-like receptors (PIRs) that serve as activating (PIR-A) and inhibitory (PIR-B) receptors on B lymphocytes, dendritic cells, and myeloid-lineage cells in mice. The monoclonal anti-PIR antibody 10.4 is shown to recognize an allelic PIR-A/PIR-B determinant on cells from BALB/c but not C57BL/6 mice. Other strains of inbred mice also can be typed on the basis of their expression of this PIR allelic determinant. Analysis of (BALB/c × C57BL/6) F1hybrid offspring indicates that PIR molecules bearing the paternal PIR allotype are expressed whereas PIR-A and PIR-B molecules bearing the maternal allotype are not. The monoallelic expression of the polymorphic PIR-A and PIR-B molecules, and possibly of their human Ig-like transcript/leukocyte Ig-like receptor/monocyte/macrophage Ig-like receptor and killer cell inhibitory receptor relatives, may influence innate and specific immune responses in outbred populations.