bluuebunny/crossref_metadata_2025 huggingface dataset:
Intrinsic near-24-h pacemaker period determines limits of circadian entrainment to a weak synchronizer in humans
Authors: Wright Kenneth P. , Hughes Rod J , Kronauer Richard E. , Dijk Derk-Jan , Czeisler Charles A.
Published: July 2002

Endogenous circadian clocks are robust regulators of physiology and behavior. Synchronization or entrainment of biological clocks to environmental time is adaptive and important for physiological homeostasis and for the proper timing of species-specific behaviors. We studied subjects in the laboratory for up to 55 days each to determine the ability to entrain the human clock to a weak circadian synchronizing stimulus [scheduled activity–rest cycle in very dim (≈1.5 lux in the angle of gaze) light–dark cycle] at three ≈24-h periods: 23.5, 24.0, and 24.6 h. These studies allowed us to test two competing hypotheses as to whether the period of the human circadian pacemaker is near to or much longer than 24 h. We report here that imposition of a sleep–wake schedule with exposure to the equivalent of candlelight during wakefulness and darkness during sleep is usually sufficient to maintain circadian entrainment to the 24-h day but not to a 23.5- or 24.6-h day. Our results demonstrate functionally that, in normally entrained sighted adults, the average intrinsic circadian period of the human biological clock is very close to 24 h. Either exposure to very dim light and/or the scheduled sleep–wake cycle itself can entrain this near-24-h intrinsic period of the human circadian pacemaker to the 24-h day.
Generation of oscillations by the p53-Mdm2 feedback loop: A theoretical and experimental study
Authors: Lev Bar-Or Ruth , Maya Ruth , Segel Lee A. , Alon Uri , Levine Arnold J. , Oren Moshe
Published: July 2002

The intracellular activity of the p53 tumor suppressor
protein is regulated through a feedback loop involving its
transcriptional target,
mdm2
. We present a simple
mathematical model suggesting that, under certain circumstances,
oscillations in p53 and Mdm2 protein levels can emerge in response to a
stress signal. A delay in p53-dependent induction of Mdm2 is predicted
to be required, albeit not sufficient, for this oscillatory behavior.
In line with the predictions of the model, oscillations of both p53 and
Mdm2 indeed occur on exposure of various cell types to ionizing
radiation. Such oscillations may allow cells to repair their DNA
without risking the irreversible consequences of continuous excessive
p53 activation.
Involvement of neurogranin in the modulation of calcium/calmodulin-dependent protein kinase II, synaptic plasticity, and spatial learning: A study with knockout mice
Authors: Pak Jhang Ho , Huang Freesia L. , Li Junfa , Balschun Detlef , Reymann Klaus G. , Chiang Chin , Westphal Heiner , Huang Kuo-Ping
Published: July 2002

Neurogranin/RC3 is a neural-specific Ca
2+
-sensitive
calmodulin (CaM)-binding protein whose CaM-binding affinity
is modulated by phosphorylation and oxidation. Here we show that
deletion of the Ng gene in mice did not result in obvious developmental
or neuroanatomical abnormalities but caused an impairment of spatial
learning and changes in hippocampal short- and long-term plasticity
(paired-pulse depression, synaptic fatigue, long-term potentiation
induction). These deficits were accompanied by a decreased basal level
of the activated Ca
2+
/CaM-dependent kinase II (CaMKII)
(≈60% of wild type). Furthermore, hippocampal slices of the mutant
mice displayed a reduced ability to generate activated CaMKII after
stimulation of protein phosphorylation and oxidation by treatments with
okadaic acid and sodium nitroprusside, respectively. These results
indicate a central role of Ng in the regulation of CaMKII activity with
decisive influences on synaptic plasticity and spatial learning.
Functional analysis of mutant and wild-type
Drosophila
origin recognition complex
Authors: Chesnokov Igor , Remus Dirk , Botchan Michael
Published: July 2002

The origin recognition complex (ORC) is the DNA replication initiator protein in eukaryotes. We have reconstituted a functional recombinant
Drosophila
ORC and compared activities of the wild-type and several mutant ORC variants.
Drosophila
ORC is an ATPase, and our studies show that the ORC1 subunit is essential for ATP hydrolysis and for ATP-dependent DNA binding. Moreover, DNA binding by ORC reduces its ATP hydrolysis activity.
In vitro
, ORC binds to chromatin in an ATP-dependent manner, and this process depends on the functional AAA
+
nucleotide-binding domain of ORC1. Mutations in the ATP-binding domain of ORC1 are unable to support cell-free DNA replication. However, mutations in the putative ATP-binding domain of either the ORC4 or ORC5 subunits do not affect either of these functions. We also provide evidence that the
Drosophila
ORC6 subunit is directly required for all of these activities and that a large pool of ORC6 is present in the cytoplasm, cytologically proximal to the cell membrane. Studies reported here provide the first functional dissection of a metazoan initiator and highlight the basic conserved and divergent features among
Drosophila
and budding yeast ORC complexes.
Tsetse immune responses and trypanosome transmission: Implications for the development of tsetse-based strategies to reduce trypanosomiasis
Authors: Hao Zhengrong , Kasumba Irene , Lehane Michael J. , Gibson Wendy C. , Kwon Johnny , Aksoy Serap
Published: July 2002

Tsetse flies are the medically and agriculturally important vectors of African trypanosomes. Information on the molecular and biochemical nature of the tsetse/trypanosome interaction is lacking. Here we describe three antimicrobial peptide genes,
attacin, defensin
, and
diptericin
, from tsetse fat body tissue obtained by subtractive cloning after immune stimulation with
Escherichia coli
and trypanosomes. Differential regulation of these genes shows the tsetse immune system can discriminate not only between molecular signals specific for bacteria and trypanosome infections but also between different life stages of trypanosomes. The presence of trypanosomes either in the hemolymph or in the gut early in the infection process does not induce transcription of
attacin
and
defensin
significantly. After parasite establishment in the gut, however, both antimicrobial genes are expressed at high levels in the fat body, apparently not affecting the viability of parasites in the midgut. Unlike other insect immune systems, the antimicrobial peptide gene
diptericin
is constitutively expressed in both fat body and gut tissue of normal and immune stimulated flies, possibly reflecting tsetse immune responses to the multiple Gram-negative symbionts it naturally harbors. When flies were immune stimulated with bacteria before receiving a trypanosome containing bloodmeal, their ability to establish infections was severely blocked, indicating that up-regulation of some immune responsive genes early in infection can act to block parasite transmission. The results are discussed in relation to transgenic approaches proposed for modulating vector competence in tsetse.
LEUNIG
, a putative transcriptional corepressor that regulates
AGAMOUS
expression during flower development
Authors: Conner Joann , Liu Zhongchi
Published: July 2002

Regulation of homeotic gene expression is critical for proper
developmental patterns in both animals and plants.
LEUNIG
is a key regulator of the
Arabidopsis
floral homeotic gene
AGAMOUS
.
Mutations in
LEUNIG
cause ectopic
AGAMOUS
mRNA expression in the outer two whorls of a flower, leading to
homeotic transformations of floral organ identity as well as loss of
floral organs. We isolated the
LEUNIG
gene by using a
map-based approach and showed that
LEUNIG
encodes a
glutamine-rich protein with seven WD repeats and is similar in motif
structure to a class of functionally related transcriptional
corepressors including Tup1 from yeast and Groucho from
Drosophila
. The nuclear localization of
LEUNIG–GFP
is consistent with a role of
LEUNIG
as a transcriptional regulator. The detection of
LEUNIG
mRNA in all floral whorls at the time of their
inception suggests that the restricted activity of
LEUNIG
in the outer two floral whorls must depend on
interactions with other spatially restricted factors or on
posttranslational regulation. Our finding suggests that both animals
and plants use similar repressor proteins to regulate critical
developmental processes.
Polycomb repression of flowering during early plant development
Authors: Kinoshita Tetsu , Harada John J. , Goldberg Robert B. , Fischer Robert L.
Published: July 2002

All plants flower late in their life cycle. For example, in
Arabidopsis
, the shoot undergoes a transition and produces reproductive flowers after the adult phase of vegetative growth. Much is known about genetic and environmental processes that control flowering time in mature plants. However, little is understood about the mechanisms that prevent plants from flowering much earlier during embryo and seedling development.
Arabidopsis embryonic flower
(
emf1
and
emf2
) mutants flower soon after germination, suggesting that a floral repression mechanism is established in wild-type plants that prevents flowering until maturity. Here, we show that polycomb group proteins play a central role in repressing flowering early in the plant life cycle. We found that mutations in the
Fertilization Independent Endosperm
(
FIE
) polycomb gene caused the seedling shoot to produce flower-like structures and organs. Flower-like structures were also generated from the hypocotyl and root, organs not associated with reproduction. Expression of floral induction and homeotic genes was derepressed in mutant embryos and seedlings. These results suggest that FIE-mediated polycomb complexes are an essential component of a floral repression mechanism established early during plant development.
Bacterial cell division protein FtsZ assembles into protofilament sheets and minirings, structural homologs of tubulin polymers.
Authors: Erickson H P , Taylor D W , Taylor K A , Bramhill D
Published: July 2002

The bacterial cell division protein FtsZ is a homolog of tubulin, but it has not been determined whether FtsZ polymers are structurally related to the microtubule lattice. In the present study, we have obtained high-resolution electron micrographs of two FtsZ polymers that show remarkable similarity to tubulin polymers. The first is a two-dimensional sheet of protofilaments with a lattice very similar to that of the microtubule wall. The second is a miniring, consisting of a single protofilament in a sharply curved, planar conformation. FtsZ minirings are very similar to tubulin rings that are formed upon disassembly of microtubules but are about half the diameter. This suggests that the curved conformation occurs at every FtsZ subunit, but in tubulin rings the conformation occurs at either beta- or alpha-tubulin subunits but not both. We conclude that the functional polymer of FtsZ in bacterial cell division is a long thin sheet of protofilaments. There is sufficient FtsZ in Escherichia coli to form a protofilament that encircles the cell 20 times. The similarity of polymers formed by FtsZ and tubulin implies that the protofilament sheet is an ancient cytoskeletal system, originally functioning in bacterial cell division and later modified to make microtubules.
A mean field model of ligand-protein interactions: implications for the structural assessment of human immunodeficiency virus type 1 protease complexes and receptor-specific binding.
Authors: Verkhivker G M , Rejto P A
Published: July 2002

We propose a general mean field model of ligand-protein interactions to determine the thermodynamic equilibrium of a system at finite temperature. The method is employed in structural assessments of two human immuno-deficiency virus type 1 protease complexes where the gross effects of protein flexibility are incorporated by utilizing a data base of crystal structures. Analysis of the energy spectra for these complexes has revealed that structural and thermo-dynamic aspects of molecular recognition can be rationalized on the basis of the extent of frustration in the binding energy landscape. In particular, the relationship between receptor-specific binding of these ligands to human immunodeficiency virus type 1 protease and a minimal frustration principle is analyzed.
Evolutionary relationships of the coelacanth, lungfishes, and tetrapods based on the 28S ribosomal RNA gene.
Authors: Zardoya R , Meyer A
Published: July 2002

The origin of land vertebrates was one of the major transitions in the history of vertebrates. Yet, despite many studies that are based on either morphology or molecules, the phylogenetic relationships among tetrapods and the other two living groups of lobe-finned fishes, the coelacanth and the lungfishes, are still unresolved and debated. Knowledge of the relationships among these lineages, which originated back in the Devonian, has profound implications for the reconstruction of the evolutionary scenario of the conquest of land. We collected the largest molecular data set on this issue so far, about 3,500 base pairs from seven species of the large 28S nuclear ribosomal gene. All phylogenetic analyses (maximum parsimony, neighbor-joining, and maximum likelihood) point toward the hypothesis that lungfishes and coelacanths form a monophyletic group and are equally closely related to land vertebrates. This evolutionary hypothesis complicates the identification of morphological or physiological preadaptations that might have permitted the common ancestor of tetrapods to colonize land. This is because the reconstruction of its ancestral conditions would be hindered by the difficulty to separate uniquely derived characters from shared derived characters in the coelacanth/lungfish and tetrapod lineages. This molecular phylogeny aids in the reconstruction of morphological evolutionary steps by providing a framework; however, only paleontological evidence can determine the sequence of morphological acquisitions that allowed lobe-finned fishes to colonize land.
A melanoma-specific VH antibody cloned from a fusion phage library of a vaccinated melanoma patient.
Authors: Cai X , Garen A
Published: July 2002

The human antimelanoma antibody V86 was cloned from a single-chain Fv molecule (scFv) fusion phage library displaying the heavy chain variable domain (VH) and light chain variable domain (VL.) repertoire of a melanoma patient immunized with genetically-modified autologous tumor cells. Previous ELISA tests for binding of the V86 fusion phage to a panel of human metastatic melanoma and carcinoma cell lines and primary cultures of normal melanocytes, endothelial, and fibroblast cells showed that measurable binding occurred only to the melanoma cells. In this communication, the strict specificity of V86 for melanoma cells was confirmed by immunohistochemical staining tests with cultured cells and frozen tissue sections. The V86 fusion phage stained melanoma cell lines but did not stain carcinoma cell lines or cultured normal cells; V86 also stained specifically the melanoma cells in sections of metastatic tissue but did not stain any of the cells in sections from normal skin, lung, and kidney or from metastatic colon and ovarian carcinomas and a benign nevus. An unexpected finding is that V86 contains a complete VH domain but only a short segment of a VL, domain, which terminates before the CDR1 region. This VL deletion resulted from the occurrence in the VL cDNA of a restriction site, which was cleaved during construction of the scFv library. Thus V86 is essentially a VH antibody. The effect of adding a VI. domain to V86 was examined by constructing scFv fusion phage libraries in which V86 was coupled to Vlambda or Vkappa domains from the original scFv library of the melanoma patient and then panning the libraries against melanoma cells to enrich for the highest affinity antibody clones. None of the V86-Vlambda clones showed significant binding to melanoma cells in ELISA tests; although binding occurred with most of the V86-Vkappa clones, it was generally weaker than the binding of V86. These results indicate that most of the VL domains in the original scFv library reduce or eliminate the affinity of V86 for melanoma cells. Accordingly, VH libraries could provide access to anti-tumor antibodies that might not be detected in scFv or Fab libraries because of the incompatibility of most randomly paired VH and VL, domains.
Restricted expression of Kaposi sarcoma-associated herpesvirus (human herpesvirus 8) genes in Kaposi sarcoma.
Authors: Zhong W , Wang H , Herndier B , Ganem D
Published: July 2002

Kaposi sarcoma (KS) is the leading neoplasm of HIV-infected patients and is also found in several HIV-negative populations. Recently, DNA sequences from a novel herpesvirus, termed KS-associated herpesvirus (KSHV), or human herpesvirus 8 (HHV-8) have been identified within KS tissue from both HIV-positive and HIV-negative cases; infection with this agent has been proposed as a possible factor in the etiology or pathogenesis of the tumor. Here we have examined the pattern of KSHV/HHV-8 gene expression in KS and find it to be highly restricted. We identify and characterize two small transcripts that represent the bulk of the virus-specific RNA transcribed from over 120 kb of the KSHV genome in infected cells. One transcript is predicted to encode a small membrane protein; the other is an unusual polyadenylylated RNA that accumulates in the nucleus to high copy number. This pattern of viral gene expression suggests that most infected cells in KS are latently infected, with lytic viral replication likely restricted to a much smaller subpopulation of cells. These findings have implications for the therapeutic utility of currently available antiviral drugs targeted against the lytic replication cycle.
Activation of RAC-protein kinase by heat shock and hyperosmolarity stress through a pathway independent of phosphatidylinositol 3-kinase.
Authors: Konishi H , Matsuzaki H , Tanaka M , Ono Y , Tokunaga C , Kuroda S , Kikkawa U
Published: July 2002

RAC protein kinase (RAC-PK), a serine/threonine protein kinase containing a pleckstrin homology (PH) domain, was activated by cellular stress such as heat shock and hyperosmolarity. Wortmannin, which is known as a potent inhibitor of phosphatidylinositol 3-kinase and normally inhibits growth factor-induced activation of RAC-PK, did not suppress heat-shock induced activation of RAC-PK, indicating that this stress-induced activation of the kinase is not mediated by phosphatidylinositol 3-kinase. The PH domain was indispensable for stress-induced activation of RAC PK. In heat-treated cells, PKC delta, a member of the protein kinase C family, was found to associate with the PH domain of RAC-PK. This PKC subspecies was phosphorylated in vitro by RAC-PK. The results suggest that RAC-PK may play a role in the cellular response to stress through its PH domain.
Transcriptionally active Stat1 is required for the antiproliferative effects of both interferon alpha and interferon gamma.
Authors: Bromberg J F , Horvath C M , Wen Z , Schreiber R D , Darnell J E
Published: July 2002

Type I (alpha, beta) and type II (gamma) interferons (IFNs) can restrict the growth of many cell types. INF-stimulated gene transcription, a key early event in IFN response, acts through the Janus kinase-signal transducers and activators of transcription pathway, in which both IFN-alpha and IFN-gamma activate the transcription factor Stat1. A cell line lacking Stat1 (U3A) was not growth-arrested by IFN-alpha or IFN-gamma, and experiments were carried out with U3A cells permanently expressing normal or various mutant forms of Stat1 protein. Only cells in which complete Stat1 activity was available (Stat1alpha) were growth-inhibited by IFN-gamma. A mutant that supports 20-30% normal transcription did not cause growth restraint. In contrast, IFN-alpha growth restraint was imposed by cells producing Stat1beta, which lacks transcriptional activation potential. This parallels earlier results showing the truncated Stat1 can function in IFN-alpha gene activation. In addition to experiments on long-term cultured cells, we also found that wild-type primary mouse embryonic fibroblasts were inhibited by IFNs, but fibroblasts from Stat1-deficient mouse embryos were not inhibited by IFNs.
Wild-type and mutant p53 differentially regulate transcription of the insulin-like growth factor I receptor gene.
Authors: Werner H , Karnieli E , Rauscher F J , LeRoith D
Published: July 2002

The insulin-like growth factor I receptor (IGF-I-R) plays a critical role in transformation events. It is highly overexpressed in most malignant tissues where it functions as an anti-apoptotic agent by enhancing cell survival. Tumor suppressor p53 is a nuclear transcription factor that blocks cell cycle progression and induces apoptosis. p53 is the most frequently mutated gene in human cancer. Cotransfection of Saos-2 (os-teosarcoma-derived cells) and RD (rhabdomyosarcoma-derived cells) cells with IGF-I-R promoter constructs driving luciferase reporter genes and with wild-type p53 expression vectors suppressed promoter activity in a dose-dependent manner. This effect of p53 is mediated at the level of transcription and it involves interaction with TBP, the TATA box-binding component of TFIID. On the other hand, three tumor-derived mutant forms of p53 (mut 143, mut 248, and mut 273) stimulated the activity of the IGF-I-R promoter and increased the levels of IGF-I-R/luciferase fusion mRNA. These results suggest that wild-type p53 has the potential to suppress the IGF-I-R promoter in the postmitotic, fully differentiated cell, thus resulting in low levels of receptor gene expression in adult tissues. Mutant versions of p53 protein, usually associated with malignant states, can derepress the IGF-I-R promoter, with ensuing mitogenic activation by locally produced or circulating IGFs.
Provirus integration into a gene encoding a ubiquitin-conjugating enzyme results in a placental defect and embryonic lethality.
Authors: Harbers K , Müller U , Grams A , Li E , Jaenisch R , Franz T
Published: July 2002

Ubiquitin-conjugating enzymes (E2 or Ubc) constitute a family of conserved proteins that play a key role in ubiquitin-dependent degradation of proteins in eukaryotes. We describe here a transgenic mouse strain where retrovirus integration into an Ubc gene, designated UbcM4, results in a recessive-lethal mutation. UbcM4 is the mouse homologue of the previously described human UbcH7 that is involved in the in vitro ubiquitination of several proteins including the tumor suppressor protein p53. The provirus is located in the first intron of the gene. When both alleles are mutated the level of steady-state mRNA is reduced by about 70%. About a third of homozygous mutant embryos die around day 11.5 of gestation. Embryos that survive that stage are growth retarded and die perinatally. The lethal phenotype is most likely caused by impairment of placenta development as this is the only organ that consistently showed pathological defects. The placental labyrinth is drastically reduced in size and vascularization is disturbed. The UbcM4 mouse mutant represents the first example in mammals of a mutation in a gene involved in ubiquitin conjugation. Its recessive-lethal phenotype demonstrates that the ubiquitin system plays an essential role during mouse development.
Torsional rigidity of single actin filaments and actin–actin bond breaking force under torsion measured directly by
in vitro
micromanipulation
Authors: Tsuda Yuri , Yasutake Hironori , Ishijima Akihiko , Yanagida Toshio
Published: July 2002

Knowledge of the elastic properties of actin filaments is crucial
for considering its role in muscle contraction, cellular motile events,
and formation of cell shape. The stiffness of actin filaments in the
directions of stretching and bending has been determined. In this
study, we have directly determined the torsional rigidity and breaking
force of single actin filaments by measuring the rotational Brownian
motion and tensile strength using optical tweezers and microneedles,
respectively. Rotational angular fluctuations of filaments supplied the
torsional rigidity as (8.0 ± 1.2) × 10
−26
Nm
2
. This value is similar to that deduced from the
longitudinal rigidity, assuming the actin filament to be a homogeneous
rod. The breaking force of the actin–actin bond was measured while
twisting a filament through various angles using microneedles. The
breaking force decreased greatly under twist, e.g., from 600–320 pN
when filaments were turned through 90°, independent of the rotational
direction. Our results indicate that an actin filament exhibits
comparable flexibility in the rotational and longitudinal directions,
but breaks more easily under torsional load.
Separation of the arterial wall from blood contact using hydrogel barriers reduces intimal thickening after balloon injury in the rat: The roles of medial and luminal factors in arterial healing
Authors: West Jennifer L. , Hubbell Jeffrey A.
Published: July 2002

The objective of this study was to clarify the relative roles of
medial versus luminal factors in the induction of thickening of the
arterial intima after balloon angioplasty injury. Platelet-derived
growth factor (PDGF) and thrombin, both associated with thrombosis, and
basic fibroblast growth factor (bFGF), stored in the arterial wall,
have been implicated in this process. To unequivocally isolate the
media from luminally derived factors, we used a 20-μm thick hydrogel
barrier that adhered firmly to the arterial wall to block thrombus
deposition after balloon-induced injury of the carotid artery of the
rat. Thrombosis, bFGF mobilization, medial repopulation, and intimal
thickening were measured. Blockade of postinjury arterial contact with
blood prevented thrombosis and dramatically inhibited both intimal
thickening and endogenous bFGF mobilization. By blocking
blood contact on the two time scales of thrombosis and of intimal
thickening, and by using local protein release to probe, by
reconstitution, the individual roles of PDGF-BB and thrombin, we were
able to conclude that a luminally derived factor other than PDGF or
thrombin is required for the initiation of cellular events leading to
intimal thickening after balloon injury in the rat. We further conclude
that a luminally derived factor is required for mobilization of medial
bFGF.
Intrinsic responses to Borna disease virus infection of the central nervous system
Authors: Morimoto Kinjiro , Hooper D. Craig , Bornhorst Annette , Corisdeo Susanne , Bette Michael , Fu Zhen Fang , Schäfer Martin K.-H. , Koprowski Hilary , Weihe Eberhard , Dietzschold Bernhard
Published: July 2002

Immune cells invading the central nervous system (CNS) in response to Borna disease virus (BDV) antigens are central to the pathogenesis of Borna disease (BD). We speculate that the response of the resident cells of the brain to infection may be involved in the sensitization and recruitment of these inflammatory cells. To separate the responses of resident cells from those of cells infiltrating from the periphery, we used dexamethasone to inhibit inflammatory reactions in BD. Treatment with dexamethasone prevented the development of clinical signs of BD, and the brains of treated animals showed no neuropathological lesions and a virtual absence of markers of inflammation, cell infiltration, or activation normally seen in the CNS of BDV-infected rats. In contrast, treatment with dexamethasone exacerbated the expression of BDV RNA, which was paralleled by a similarly elevated expression of mRNAs foregr-1, c-fos, and c-jun. Furthermore, dexamethasone failed to inhibit the increase in expression of mRNAs for tumor necrosis factor α, macrophage inflammatory protein 1β, interleukin 6, andmob-1, which occurs in the CNS of animals infected with BDV. Our findings suggest that these genes, encoding transcription factors, chemokines, and proinflammatory cytokines, might be directly activated in CNS resident cells by BDV. This result supports the hypothesis that the initial phase of the inflammatory response to BDV infection in the brain may be dependent upon virus-induced activation of CNS resident cells.
Transcription-induced mutations: Increase in C to T mutations in the nontranscribed strand during transcription in
Escherichia coli
Authors: Beletskii A. , Bhagwat Ashok S.
Published: July 2002

Cytosines in single-stranded DNA deaminate to uracils at 140 times
the rate for cytosines in double-stranded DNA. If resulting uracils are
not replaced with cytosine, C to T mutations occur. These facts suggest
that cellular processes such as transcription that create
single-stranded DNA should promote C to T mutations. We tested this
hypothesis with the
Escherichia coli tac
promoter and
found that induction of transcription causes ≈4-fold increase in the
frequency of C to U or 5-methylcytosine to T deaminations in the
nontranscribed strand. Excess mutations caused by C to U deaminations
were reduced, but not eliminated, by uracil-DNA glycosylase. Similarly,
mutations caused by 5-methylcytosine to T deaminations were only
partially reduced by the very short-patch repair process in
E.
coli
. These effects are unlikely to be caused by differential
repair of the two strands, and our results suggest that all actively
transcribed genes in
E. coli
should acquire more C to T
mutations in the nontranscribed strand.
Brain monoamine oxidase A inhibition in cigarette smokers
Authors: Fowler Joanna S. , Volkow Nora D. , Wang Gene-Jack , Pappas Naomi , Logan Jean , Shea Colleen , Alexoff David , MacGregor Robert R. , Schlyer David J. , Zezulkova Ivana , Wolf Alfred P.
Published: July 2002

Several studies have documented a strong association between
smoking and depression. Because cigarette smoke has been reported to
inhibit monoamine oxidase (MAO) A
in vitro
and in
animals and because MAO A inhibitors are effective antidepressants, we
tested the hypothesis that MAO A would be reduced in the brain of
cigarette smokers. We compared brain MAO A in 15 nonsmokers and 16
current smokers with [
11
C]clorgyline and positron
emission tomography (PET). Four of the nonsmokers were also treated
with the antidepressant MAO inhibitor drug, tranylcypromine (10
mg/day for 3 days) after the baseline PET scan and then rescanned to
assess the sensitivity of [
11
C]clorgyline binding to MAO
inhibition. MAO A levels were quantified by using the model term
λk
3
which is a function of brain MAO A concentration.
Smokers had significantly lower brain MAO A than nonsmokers in all
brain regions examined (average reduction, 28%). The mean
λ
k
3
values for the whole brain were 0.18
± 0.04 and 0.13 ± 0.03 cc
brain
(ml
plasma
)
−1
min
−1
for nonsmokers
and smokers, respectively;
P
< 0.0003).
Tranylcypromine treatment reduced λ
k
3
by
an average of 58% for the different brain regions. Our results show
that tobacco smoke exposure is associated with a marked reduction in
brain MAO A, and this reduction is about half of that produced by a
brief treatment with tranylcypromine. This suggests that MAO A
inhibition needs to be considered as a potential contributing variable
in the high rate of smoking in depression and in the development of
more effective strategies for smoking cessation.
Molecular ordering of the Fas-apoptotic pathway: The Fas/APO-1 protease Mch5 is a CrmA-inhibitable protease that activates multiple Ced-3/ICE-like cysteine proteases
Authors: Srinivasula Srinivasa M. , Ahmad Manzoor , Fernandes-Alnemri Teresa , Litwack Gerald , Alnemri Emad S.
Published: July 2002

The Fas/APO-1-receptor associated cysteine protease Mch5
(MACH/FLICE) is believed to be the enzyme responsible for activating
a protease cascade after Fas-receptor ligation, leading to cell death.
The Fas-apoptotic pathway is potently inhibited by the cowpox
serpin CrmA, suggesting that Mch5 could be the target of this serpin.
Bacterial expression of proMch5 generated a mature enzyme composed of
two subunits, which are derived from the precursor proenzyme by
processing at Asp-227, Asp-233, Asp-391, and Asp-401. We demonstrate
that recombinant Mch5 is able to process/activate all known
ICE/Ced-3-like cysteine proteases and is potently inhibited by CrmA.
This contrasts with the observation that Mch4, the second FADD-related
cysteine protease that is also able to process/activate all known
ICE/Ced-3-like cysteine proteases, is poorly inhibited by CrmA. These
data suggest that Mch5 is the most upstream protease that receives the
activation signal from the Fas-receptor to initiate the
apoptotic protease cascade that leads to activation of ICE-like
proteases (TX, ICE, and ICE-relIII), Ced-3-like proteases (CPP32, Mch2,
Mch3, Mch4, and Mch6), and the ICH-1 protease. On the other hand, Mch4
could be a second upstream protease that is responsible for activation
of the same protease cascade in CrmA-insensitive apoptotic
pathways.
The N-terminal domain of a K
+
channel β subunit increases the rate of C-type inactivation from the cytoplasmic side of the channel
Authors: Morales Michael J. , Wee Jonny O. , Wang Shimin , Strauss Harold C. , Rasmusson Randall L.
Published: July 2002

Voltage-gated K
+
channels are complexes of
membrane-bound, ion-conducting α and cytoplasmic ancillary (β)
subunits. The primary physiologic effect of coexpression of α and β
subunits is to increase the intrinsic rate of inactivation of the α
subunit. For one β subunit, Kvβ1.1, inactivation is enhanced
through an N-type mechanism. A second β subunit, Kvβ1.2, has been
shown to increase inactivation, but through a distinct mechanism. Here
we show that the degree of enhancement of Kvβ1.2 inactivation is
dependent on the amino acid composition in the pore mouth of the α
subunit and the concentration of extracellular K
+
.
Experimental conditions that promote C-type inactivation also enhance
the stimulation of inactivation by Kvβ1.2, showing that this β
subunit directly stimulates C-type inactivation. Chimeric constructs
containing just the nonconserved N-terminal region of Kvβ1.2 fused
with an α subunit behave in a similar fashion to coexpressed Kvβ1.2
and α subunit. This shows that it is the N-terminal domain of
Kvβ1.2 that mediates the increase in C-type inactivation from the
cytoplasmic side of the pore. We propose a model whereby the N terminus
of Kvβ1.2 acts as a weakly binding “ball” domain that
associates with the intracellular vestibule of the α subunit to
effect a conformational change leading to enhancement of C-type
inactivation.
Effect of β-amyloid block of the fast-inactivating K
+
channel on intracellular Ca
2+
and excitability in a modeled neuron
Authors: Good Theresa A. , Murphy Regina M.
Published: July 2002

β-Amyloid peptide (Aβ), one of the primary protein components
of senile plaques found in Alzheimer disease, is believed to be toxic
to neurons by a mechanism that may involve loss of intracellular
calcium regulation. We have previously shown that Aβ blocks the
fast-inactivating potassium (A) current. In this work, we show, through
the use of a mathematical model, that the Aβ-mediated block of the A
current could result in increased intracellular calcium levels and
increased membrane excitability, both of which have been observed
in vitro
upon acute exposure to Aβ. Simulation results
are compared with experimental data from the literature; the
simulations quantitatively capture the observed concentration
dependence of the neuronal response and the level of increase in
intracellular calcium.
Cell surface expression of the epithelial Na channel and a mutant causing Liddle syndrome: A quantitative approach
Authors: Firsov Dmitri , Schild Laurent , Gautschi Ivan , Mérillat Anne-Marie , Schneeberger Estelle , Rossier Bernard C.
Published: July 2002

The epithelial amiloride-sensitive sodium channel (ENaC) controls
transepithelial Na
+
movement in
Na
+
-transporting epithelia and is associated with Liddle
syndrome, an autosomal dominant form of salt-sensitive hypertension.
Detailed analysis of ENaC channel properties and the functional
consequences of mutations causing Liddle syndrome has been, so far,
limited by lack of a method allowing specific and quantitative
detection of cell-surface-expressed ENaC. We have developed a
quantitative assay based on the binding of
125
I-labeled
M
2
anti-FLAG monoclonal antibody (M
2
Ab*)
directed against a FLAG reporter epitope introduced in the
extracellular loop of each of the α, β, and γ ENaC subunits.
Insertion of the FLAG epitope into ENaC sequences did not change its
functional and pharmacological properties. The binding specificity and
affinity (
K
d
= 3 nM) allowed us to correlate
in individual
Xenopus
oocytes the macroscopic
amiloride-sensitive sodium current (I
Na
) with the number of
ENaC wild-type and mutant subunits expressed at the cell surface. These
experiments demonstrate that: (
i
) only heteromultimeric
channels made of α, β, and γ ENaC subunits are maximally and
efficiently expressed at the cell surface; (
ii
) the
overall ENaC open probability is one order of magnitude lower than
previously observed in single-channel recordings; (
iii
)
the mutation causing Liddle syndrome (β R564stop) enhances channel
activity by two mechanisms, i.e., by increasing ENaC cell surface
expression and by changing channel open probability. This quantitative
approach provides new insights on the molecular mechanisms underlying
one form of salt-sensitive hypertension.
A new selenoprotein from human lung adenocarcinoma cells: purification, properties, and thioredoxin reductase activity.
Authors: Tamura T , Stadtman T C
Published: July 2002

We report the isolation and characterization of a new selenoprotein from a human lung adenocarcinoma cell line, NCI-H441. Cells were grown in RPMI-1640 medium containing 10% (vol/vol) fetal bovine serum and 0.1 microM [75Se]selenite. A 75Se-labeled protein was isolated from sonic extracts of the cells by chromatography on DE-23, phenyl-Sepharose, heparin-agarose, and butyl-Sepharose. The protein, a homodimer of 57-kDa subunits, was shown to contain selenium in the form of selenocysteine; hydrolysis of the protein alkylated with either iodoacetate or 3-bromopropionate yielded Se-carboxymethyl-selenocysteine or Se-carboxyethyl-selenocysteine, respectively. The selenoprotein showed two isoelectric points at pH 5.2 and pH 5.3. It was distinguished from selenoprotein P by N-glycosidase assay and by the periodate-dansylhydrazine test, which indicated no detectable amounts of glycosyl groups on the protein. The selenoprotein contains FAD as a prosthetic group and catalyzes NADPH-dependent reduction of 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), and reduction of insulin in the presence of thioredoxin (Trx). The specific activity was determined to be 31 units/mg by DTNB assay. Apparent Km values for DTNB, Escherichia coli Trx, and rat Trx were 116, 34, and 3.7 microM, respectively. DTNB reduction was inhibited by 0.2 mM arsenite. Although the subunit composition and catalytic properties are similar to those of mammalian thioredoxin reductase (TR), the human lung selenoprotein failed to react with anti-rat liver TR polyclonal antibody in immunoblot assays. The selenocysteine-containing TR from the adenocarcinoma cells may be a variant form distinct from rat liver TR.
Translation initiation of ornithine decarboxylase and nucleocytoplasmic transport of cyclin D1 mRNA are increased in cells overexpressing eukaryotic initiation factor 4E.
Authors: Rousseau D , Kaspar R , Rosenwald I , Gehrke L , Sonenberg N
Published: July 2002

The structure of m7GpppN (where N is any nucleotide), termed cap, is present at the 5' end of all eukaryotic cellular mRNAs (except organellar). The eukaryotic initiation factor 4E (eIF-4E) binds to the cap and facilitates the formation of translation initiation complexes. eIF-4E is implicated in control of cell growth, as its overexpression causes malignant transformation of rodent cells and deregulates HeLa cell growth. It was suggested that overexpression of eIF-4E results in the enhanced translation of poorly translated mRNAs that encode growth-promoting proteins. Indeed, enhanced expression of several proteins, including cyclin D1 and ornithine decarboxylase (ODC), was documented in eIF-4E-overexpressing NTH 3T3 cells. However, the mechanism underlying this increase has not been elucidated. Here, we studied the mode by which eIF-4E increases the expression of cyclin D1 and ODC. We show that the increase in the amount of cyclin D1 and ODC is directly proportional to the degree of eIF-4E overexpression. Two mechanisms, which are not mutually exclusive, are responsible for the increase. In eIF-4E-overexpressing cells the rate of translation initiation of ODC mRNA was increased inasmuch as the mRNA sedimented with heavier polysomes. For cyclin D1 mRNA, translation initiation was not increased, but rather its amount in the cytoplasm increased, without a significant increase in total mRNA. Whereas, in the parental NIH 3T3 cell line, a large proportion of the cyclin D1 mRNA was confined to the nucleus, in eIF-4E-overexpressing cells the vast majority of the mRNA was present in the cytoplasm. These results indicate that eIF-4E affects directly or indirectly mRNA nucleocytoplasmic transport, in addition to its role in translation initiation.
Cleavage of actin by interleukin 1 beta-converting enzyme to reverse DNase I inhibition.
Authors: Kayalar C , Ord T , Testa M P , Zhong L T , Bredesen D E
Published: July 2002

Three of the predominant features of apoptosis are internucleosomal DNA fragmentation, plasma membrane bleb formation, and retraction of cell processes. We demonstrate that actin is a substrate for the proapoptotic cysteine protease interleukin 1beta-converting enzyme. Actin cleaved by interleukin 1beta-converting enzyme can neither inhibit DNase I nor polymerize to its filamentous form as effectively as intact actin. These findings suggest a mechanism for the coordination of the proteolytic, endonucleolytic, and morphogenetic aspects of apoptosis.
COX-2, a synaptically induced enzyme, is expressed by excitatory neurons at postsynaptic sites in rat cerebral cortex.
Authors: Kaufmann W E , Worley P F , Pegg J , Bremer M , Isakson P
Published: July 2002

Postnatal development and adult function of the central nervous system are dependent on the capacity of neurons to effect long-term changes of specific properties in response to neural activity. This neuronal response has been demonstrated to be tightly correlated with the expression of a set of regulatory genes which include transcription factors as well as molecules that can directly modify cellular signaling. It is hypothesized that these proteins play a role in activity-dependent response. Previously, we described the expression and regulation in brain of an inducible form of prostaglandin synthase/cyclooxygenase, termed COX-2. COX-2 is a rate-limiting enzyme in prostanoid synthesis and its expression is rapidly regulated in developing and adult forebrain by physiological synaptic activity. Here we demonstrate that COX-2 immunoreactivity is selectively expressed in a subpopulation of excitatory neurons in neo-and allocortices, hippocampus, and amygdala and is compartmentalized to dendritic arborizations. Moreover, COX-2 immunoreactivity is present in dendritic spines, which are specialized structures involved in synaptic signaling. The developmental profile of COX-2 expression in dendrites follows well known histogenetic gradients and coincides with the critical period for activity-dependent synaptic remodeling. These results suggest that COX-2, and its diffusible prostanoid products, may play a role in postsynaptic signaling of excitatory neurons in cortex and associated structures.
Visualization of the vesicular acetylcholine transporter in cholinergic nerve terminals and its targeting to a specific population of small synaptic vesicles.
Authors: Weihe E , Tao-Cheng J H , Schäfer M K , Erickson J D , Eiden L E
Published: July 2002

Immunohistochemical visualization of the rat vesicular acetylcholine transporter (VAChT) in cholinergic neurons and nerve terminals has been compared to that for choline acetyltransferase (ChAT), heretofore the most specific marker for cholinergic neurons. VAChT-positive cell bodies were visualized in cerebral cortex, basal forebrain, medial habenula, striatum, brain stem, and spinal cord by using a polyclonal anti-VAChT antiserum. VAChT-immuno-reactive fibers and terminals were also visualized in these regions and in hippocampus, at neuromuscular junctions within skeletal muscle, and in sympathetic and parasympathetic autonomic ganglia and target tissues. Cholinergic nerve terminals contain more VAChT than ChAT immunoreactivity after routine fixation, consistent with a concentration of VAChT within terminal neuronal arborizations in which secretory vesicles are clustered. These include VAChT-positive terminals of the median eminence or the hypothalamus, not observed with ChAT antiserum after routine fixation. Subcellular localization of VAChT in specific organelles in neuronal cells was examined by immunoelectron microscopy in a rat neuronal cell line (PC 12-c4) expressing VAChT as well as the endocrine and neuronal forms of the vesicular monoamine transporters (VMAT1 and VMAT2). VAChT is targeted to small synaptic vesicles, while VMAT1 is found mainly but not exclusively on large dense-core vesicles. VMAT2 is found on large dense-core vesicles but not on the small synaptic vesicles that contain VAChT in PC12-c4 cells, despite the presence of VMAT2 immunoreactivity in central and peripheral nerve terminals known to contain monoamines in small synaptic vesicles. Thus, VAChT and VMAT2 may be specific markers for "cholinergic" and "adrenergic" small synaptic vesicles, with the latter not expressed in nonstimulated neuronally differentiated PC12-c4 cells.
A multipurpose transposon system for analyzing protein production, localization, and function in
Saccharomyces cerevisiae
Authors: Ross-Macdonald Petra , Sheehan Amy , Roeder G. Shirleen , Snyder Michael
Published: July 2002

Analysis of the function of a particular gene product typically
involves determining the expression profile of the gene, the
subcellular location of the protein, and the phenotype of a null strain
lacking the protein. Conditional alleles of the gene are often created
as an additional tool. We have developed a multifunctional,
transposon-based system that simultaneously generates constructs for
all the above analyses and is suitable for mutagenesis of any given
Saccharomyces cerevisiae
gene. Depending on the
transposon used, the yeast gene is fused to a coding region for
β-galactosidase or green fluorescent protein. Gene expression can
therefore be monitored by chemical or fluorescence assays. The
transposons create insertion mutations in the target gene, allowing
phenotypic analysis. The transposon can be reduced by
cre–lox
site-specific recombination to a smaller
element that leaves an epitope tag inserted in the encoded protein. In
addition to its utility for a variety of immunodetection purposes, the
epitope tag element also has the potential to create conditional
alleles of the target gene. We demonstrate these features of the
transposons by mutagenesis of the
SPA2
,
ARP100
,
SER1
, and
BDF1
genes.
Sialadenitis histologically resembling Sjögren syndrome in mice transgenic for hepatitis C virus envelope genes
Authors: Koike Kazuhiko , Moriya Kyoji , Ishibashi Kotaro , Yotsuyanagi Hiroshi , Shintani Yoshizumi , Fujie Hajime , Kurokawa Kiyoshi , Matsuura Yoshiharu , Miyamura Tatsuo
Published: July 2002

Hepatitis C virus (HCV), a major causative agent of non-A, non-B chronic hepatitis, is also suggested to be associated with extrahepatic manifestations such as mixed cryoglobulinemia and glomerulonephritis. Two independent lines of transgenic mice carrying the HCV envelope genes have been shown previously to express the HCV envelope proteins in organs, including the liver and salivary glands, which results in no pathological changes in the liver. Further analysis of these animals now has revealed that they develop an exocrinopathy involving the salivary and lachrymal glands. This pathology resembles Sjögren syndrome, which also is suggested to have a possible association with chronic hepatitis C. These observations suggest that HCV might be involved in the pathogenesis of sialadenitis in humans and that this transgenic mouse system would be a good animal model for the study of HCV infection.