bluuebunny/crossref_metadata_2025 huggingface dataset:
Bimodal regulation of
mPeriod
promoters by CREB-dependent signaling and CLOCK/BMAL1 activity
Authors: Travnickova-Bendova Zdenka , Cermakian Nicolas , Reppert Steven M. , Sassone-Corsi Paolo
Published: July 2002

Circadian rhythmicity in mammals is under the control of a molecular pacemaker constituted of clock gene products organized in transcriptional autoregulatory loops. Phase resetting of the clock in response to light involves dynamic changes in the expression of several clock genes. The molecular pathways used by light to influence pacemaker-driven oscillation of clock genes remain poorly understood. We explored the functional integration of both light- and clock-responsive transcriptional regulation at the promoter level of the
Period
(
Per
) genes. Three
Per
genes exist in the mouse. Whereas
mPer1
and
mPer2
are light-inducible in clock neurons of the hypothalamic suprachiasmatic nucleus,
mPer3
is not. We have studied the promoter structure of the three
mPer
genes and compared their regulation. All three
mPer
promoters contain E-boxes and respond to the CLOCK/brain and muscle aryl hydrocarbon receptor nuclear translocator (ARNT)-like protein 1 (BMAL1) heterodimer. On the other hand, only
mPer1
and
mPer2
promoters contain bona fide cAMP-responsive elements (CREs) that bind CRE-binding protein (CREB) from suprachiasmatic nucleus protein extracts. The
mPer1
promoter is responsive to synergistic activation of the cAMP and mitogen-activated protein kinase pathways, a physiological response that requires integrity of the CRE. In contrast, activation of
mPer
promoters by CLOCK/BMAL1 occurs regardless of an intact CRE. Altogether, these results constitute strong evidence that CREB acts as a pivotal endpoint of signaling pathways for the regulation of
mPer
genes. Our results reveal that signaling-dependent activation of
mPer
genes is distinct from the CLOCK/BMAL1-driven transcription required within the clock feedback loop.
Reproductive skew in the polygynandrous acorn woodpecker
Authors: Haydock Joseph , Koenig Walter D.
Published: July 2002

Reproductive skew models, which focus on the degree to which reproduction is shared equally (low skew) or monopolized by a single individual (high skew) within groups, have been heralded as providing a general unifying framework for understanding the factors determining social evolution. Here, we test the ability of optimal skew, or “transactional,” models, which predict the level of skew necessary to promote stable associations of dominants and subordinates, rather than independent breeding, to predict reproductive partitioning in the acorn woodpecker (Melanerpes formicivorus). This species provides a key test case because only a few vertebrates exhibit polygynandry (multiple breeders of both sexes within a group). Contrary to the predictions of the models, joint-nesting females share reproduction more equitably than expected, apparently because egg destruction and the inability of females to defend their eggs from cobreeders eliminate any possibility for one female to control reproduction. For males, however, reproductive skew is high, with the most successful male siring over three times as many young as the next most successful male. Although this result is consistent with optimal skew models, other aspects of male behavior are not; in particular, the reproductively most successful male frequently switches between nests produced by the same set of cobreeders, and we were unable to detect any phenotypic correlate of success. These results are consistent with an alternative null model in which cobreeder males have equal chance of paternity, but paternity of offspring within broods is nonindependent as a consequence of female, rather than male, control.
Solution structure of DFF40 and DFF45 N-terminal domain complex and mutual chaperone activity of DFF40 and DFF45
Authors: Zhou Pei , Lugovskoy Alexey A. , McCarty John S. , Li Peng , Wagner Gerhard
Published: July 2002

Apoptotic DNA fragmentation is mediated by a
caspase-activated DNA fragmentation factor (DFF)40. Expression and
folding of DFF40 require the presence of DFF45, which also acts as a
nuclease inhibitor before DFF40 activation by execution caspases. The
N-terminal domains (NTDs) of both proteins are homologous, and their
interaction plays a key role in the proper functioning of this
two-component system. Here we report that the NTD of DFF45 alone is
unstructured in solution, and its folding is induced upon binding to
DFF40 NTD. Therefore, folding of both proteins regulates the formation
of the DFF40/DFF45 complex. The solution structure of the
heterodimeric complex between NTDs of DFF40 and DFF45 reported here
shows that the mutual chaperoning includes the formation of an
extensive network of intermolecular interactions that bury a
hydrophobic cluster inside the interface, surrounded by intermolecular
salt bridges.
Temperature and length scale dependence of hydrophobic effects and their possible implications for protein folding
Authors: Huang David M. , Chandler David
Published: July 2002

The Lum–Chandler–Weeks theory of hydrophobicity [Lum, K., Chandler, D. & Weeks, J. D. (1999)
J. Phys. Chem.
103, 4570–4577] is applied to treat the temperature dependence of hydrophobic solvation in water. The application illustrates how the temperature dependence for hydrophobic surfaces extending less than 1 nm differs significantly from that for surfaces extending more than 1 nm. The latter is the result of water depletion, a collective effect, that appears at length scales of 1 nm and larger. Because of the contrasting behaviors at small and large length scales, hydrophobicity by itself can explain the variable behavior of entropies of protein folding.
In vivo
requirement for RecJ, ExoVII, ExoI, and ExoX in methyl-directed mismatch repair
Authors: Burdett Vickers , Baitinger Celia , Viswanathan Mohan , Lovett Susan T. , Modrich Paul
Published: July 2002

Biochemical studies with model DNA heteroduplexes have implicated
RecJ exonuclease, exonuclease VII, exonuclease I, and exonuclease X in
Escherichia coli
methyl-directed mismatch correction.
However, strains deficient in the four exonucleases display only a
modest increase in mutation rate, raising questions concerning
involvement of these activities in mismatch repair
in
vivo
. The quadruple mutant deficient in the four exonucleases,
as well as the triple mutant deficient in RecJ exonuclease, exonuclease
VII, and exonuclease I, grow poorly in the presence of the base
analogue 2-aminopurine, and exposure to the base analogue results in
filament formation, indicative of induction of SOS DNA damage response.
The growth defect and filamentation phenotypes associated with
2-aminopurine exposure are effectively suppressed by null mutations in
mutH
,
mutL
,
mutS
, or
uvrD/mutU
, which encode activities that act upstream
of the four exonucleases in the mechanism for the methyl-directed
reaction that has been proposed based on
in vitro
studies. The quadruple exonuclease mutant is also cold-sensitive,
having a severe growth defect at 30°C. This phenotype is suppressed
by a
uvrD/mutU
defect, and partially suppressed by
mutH
,
mutL
, or
mutS
mutations. These observations confirm involvement of the four
exonucleases in methyl-directed mismatch repair
in vivo
and suggest that the low mutability of exonuclease-deficient strains is
a consequence of under recovery of mutants due to a reduction in
viability and/or chromosome loss associated with activation of the
mismatch repair system in the absence of RecJ exonuclease, exonuclease
VII, exonuclease I, and exonuclease X.
Preferential cleavage of plasmid-based R-loops and D-loops by
Drosophila
topoisomerase IIIβ
Authors: Wilson-Sali Tina , Hsieh Tao-shih
Published: July 2002

The topoisomerase (topo) III enzymes are found in organisms ranging from bacteria to humans, yet the precise cellular function of these enzymes remains to be determined. We previously found that
Drosophila
topo IIIβ can relax plasmid DNA only if the DNA is first hypernegatively supercoiled. To investigate the possibility that topo IIIβ requires a single-stranded region for its relaxation activity, we formed R-loops and D-loops in plasmids. In addition to containing a single-stranded region, these R-loops and D-loops have the advantage of being covalently closed and supercoiled, thus allowing us to assay for supercoil relaxation. We found that topo IIIβ preferentially cleaves, rather than relaxes, these substrates. The cleavage of the R-loops and D-loops, which is primarily in the form of nicking, occurs to a greater extent at a temperature that is lower than the optimal temperature for relaxation of hypernegatively supercoiled plasmid. In addition, the cleavage can be readily reversed by high salt or high temperature, and the products fail to enter the gel in the absence of proteinase K treatment and are not observed with an active-site Y332F mutant of topo IIIβ, indicating that the cleavage is mediated by a topoisomerase. We mapped the cleavage to the unpaired strand within the loop region and found that the cleavage occurs along the length of the unpaired strand. These studies suggest that the topo III enzyme behaves as a structure-specific endonuclease
in vivo
, providing a reversible DNA cleavage activity that is specific for unpaired regions in the DNA.
Brain activity evidence for recognition without recollection after early hippocampal damage
Authors: Düzel E. , Vargha-Khadem F. , Heinze H. J. , Mishkin M.
Published: July 2002

Amnesic patients with early and seemingly isolated hippocampal injury show relatively normal recognition memory scores. The cognitive profile of these patients raises the possibility that this recognition performance is maintained mainly by stimulus familiarity in the absence of recollection of contextual information. Here we report electrophysiological data on the status of recognition memory in one of the patients, Jon. Jon's recognition of studied words lacks the event-related potential (ERP) index of recollection, viz., an increase in the late positive component (500–700 ms), under conditions that elicit it reliably in normal subjects. On the other hand, a decrease of the ERP amplitude between 300 and 500 ms, also reliably found in normal subjects, is well preserved. This so-called N400 effect has been linked to stimulus familiarity in previous ERP studies of recognition memory. In Jon, this link is supported by the finding that his recognized and unrecognized studied words evoked topographically distinct ERP effects in the N400 time window. These data suggest that recollection is more dependent on the hippocampal formation than is familiarity, consistent with the view that the hippocampal formation plays a special role in episodic memory, for which recollection is so critical.
Substrate-specific regulation of the ribosome– translocon junction by N-terminal signal sequences
Authors: Rutkowski D. Thomas , Lingappa Vishwanath R. , Hegde Ramanujan S.
Published: July 2002

Amino-terminal signal sequences target nascent secretory and membrane proteins to the endoplasmic reticulum for translocation. Subsequent interactions between the signal sequence and components of the translocation machinery at the endoplasmic reticulum are thought to be important for the productive engagement of the translocon by the ribosome-nascent chain complex. However, it is not clear whether all signal sequences carry out these posttargeting steps identically, or if there are differences in the interactions directed by one signal sequence versus another. In this study, we find substantial differences in the ability of signal sequences from different substrates to mediate closure of the ribosome–translocon junction early in translocation. We also show that these differences in some cases necessitate functional coordination between the signal sequence and mature domain for faithful translocation. Accordingly, the translocation of some proteins is sensitive to replacement of their signal sequences. In a particularly dramatic example, the topology of the prion protein was found to depend highly on the choice of signal sequence used to direct its translocation. Taken together, our results reveal an unanticipated degree of substrate-specific functionality encoded in N-terminal signal sequences.
Reelin controls position of autonomic neurons in the spinal cord
Authors: Yip Joseph W. , Yip Yee Ping L. , Nakajima Kazunori , Capriotti Christine
Published: July 2002

Mutation of the reeler gene (
Reln
) disrupts neuronal migration in several brain regions and gives rise to functional deficits such as ataxic gait and trembling in the reeler mutant mouse. Thus, the
Reln
product, reelin, is thought to control cell–cell interactions critical for cell positioning in the brain. Although an abundance of reelin transcript is found in the embryonic spinal cord [Ikeda, Y. & Terashima, T. (1997)
Dev. Dyn.
210, 157–172; Schiffmann, S. N., Bernier, B. & Goffinet, A. M. (1997)
Eur. J. Neurosci.
9, 1055–1071], it is generally thought that neuronal migration in the spinal cord is not affected by reelin. Here, however, we show that migration of sympathetic preganglionic neurons in the spinal cord is affected by reelin. This study thus indicates that reelin affects neuronal migration outside of the brain. Moreover, the relationship between reelin and migrating preganglionic neurons suggests that reelin acts as a barrier to neuronal migration.
The spliceosomal snRNP core complex ofTrypanosoma brucei: Cloning and functional analysis reveals seven Sm protein constituents
Authors: Palfi Zsofia , Lücke Stephan , Lahm Hans-Werner , Lane William S. , Kruft Volker , Bragado-Nilsson Elisabeth , Séraphin Bertrand , Bindereif Albrecht
Published: July 2002

Each of the trypanosome small nuclear ribonucleoproteins (snRNPs) U2, U4/U6, and U5, as well as the spliced leader (SL) RNP, contains a core of common proteins, which we have previously identified. This core is unusual because it is not recognized by anti-Sm Abs and it associates with an Sm-related sequence in the trypanosome small nuclear RNAs (snRNAs). Using peptide sequences derived from affinity-purified U2 snRNP proteins, we have cloned cDNAs for five common proteins of 8.5, 10, 12.5, 14, and 15 kDa ofTrypanosoma bruceiand identified them as Sm proteins SmF (8.5 kDa), -E (10 kDa), -D1 (12.5 kDa), -G (14 kDa), and -D2 (15 kDa), respectively. Furthermore, we found the trypanosome SmB (T. brucei) and SmD3 (Trypanosoma cruzi) homologues through database searches, thus completing a set of seven canonical Sm proteins. Sequence comparisons of the trypanosome proteins revealed several deviations in highly conserved positions from the Sm consensus motif. We have identified a network of specific heterodimeric and -trimeric Sm protein interactionsin vitro. These results are summarized in a model of the trypanosome Sm core, which argues for a strong conservation of the Sm particle structure. The conservation extends also to the functional level, because at least one trypanosome Sm protein, SmG, was able to specifically complement a corresponding mutation in yeast.
DNA bending by an adenine–thymine tract and its role in gene regulation
Authors: Hizver Jenny , Rozenberg Haim , Frolow Felix , Rabinovich Dov , Shakked Zippora
Published: July 2002

To gain insight into the structural basis of DNA bending by adenine–thymine tracts (A-tracts) and their role in DNA recognition by gene-regulatory proteins, we have determined the crystal structure of the high-affinity DNA target of the cancer-associated human papillomavirus E2 protein. The three independent B-DNA molecules of the crystal structure determined at 2.2-Å resolution are examples of A-tract-containing helices where the global direction and magnitude of curvature are in accord with solution data, thereby providing insights, at the base pair level, into the mechanism of DNA bending by such sequence motifs. A comparative analysis of E2–DNA conformations with respect to other structural and biochemical studies demonstrates that (i) the A-tract structure of the core region, which is not contacted by the protein, is critical for the formation of the high-affinity sequence-specific protein–DNA complex, and (ii) differential binding affinity is regulated by the intrinsic structure and deformability encoded in the base sequence of the DNA target.
Colonization of America byDrosophila subobscura: Heterotic effect of chromosomal arrangements revealed by the persistence of lethal genes
Authors: Mestres F. , Balanyà J. , Arenas C. , Solé E. , Serra L.
Published: July 2002

About 20 years agoDrosophila subobscura, a native Palearctic species, colonized both North and South America. In Palearctic populations lethal genes are not associated in general with particular chromosomal arrangements. In colonizing populations they are not randomly distributed and usually are associated to a different degree with chromosomal arrangements caused by the founder event. The persistence of two lethal genes in the colonizing populations, one completely associated with the O5inversion and the other partially associated with the O3+4+7arrangement, has been analyzed. In all populations studied (five North American and six South American) the observed frequency of the lethal gene completely associated with the O5inversion is higher than expected, the difference being statistically significant in all South American and one North American populations. The observed frequency of the lethal gene partially associated with the O3+4+7arrangement is also significantly higher than expected. Taking into account that the O5inversion exhibits significant latitudinal clines both in North and South America, an overdominant model favoring the heterokaryotypes seems to be in operation. From this model, a polynomial expression has been developed that allows us to estimate the relative fitness and the coefficient of selection against all karyotypes not carrying the O5inversion. The relative fitness of the O5heterokaryotypes is higher in South American than in North American populations. Furthermore, the observed frequencies of the lethal genes studied are in general very close to those of the equilibrium. This case is an outstanding demonstration in nature of an heterotic effect of chromosomal segments associated with lethal genes on a large geographic scale.
Controlling potassium channel activities: Interplay between the membrane and intracellular factors
Authors: Yi B. Alexander , Minor Daniel L. , Lin Yu-Fung , Jan Yuh Nung , Jan Lily Yeh
Published: July 2002

Neural signaling is based on the regulated timing and extent of
channel opening; therefore, it is important to understand how ion
channels open and close in response to neurotransmitters and
intracellular messengers. Here, we examine this question for potassium
channels, an extraordinarily diverse group of ion channels.
Voltage-gated potassium (Kv) channels control action-potential
waveforms and neuronal firing patterns by opening and closing in
response to membrane-potential changes. These effects can be strongly
modulated by cytoplasmic factors such as kinases, phosphatases, and
small GTPases. A Kv α subunit contains six transmembrane segments,
including an intrinsic voltage sensor. In contrast, inwardly rectifying
potassium (Kir) channels have just two transmembrane segments in each
of its four pore-lining α subunits. A variety of intracellular second
messengers mediate transmitter and metabolic regulation of Kir
channels. For example, Kir3 (GIRK) channels open on binding to the G
protein βγ subunits, thereby mediating slow inhibitory postsynaptic
potentials in the brain. Our structure-based functional analysis on the
cytoplasmic N-terminal tetramerization domain T1 of the voltage-gated
channel, Kv1.2, uncovered a new function for this domain, modulation of
voltage gating, and suggested a possible means of communication between
second messenger pathways and Kv channels. A yeast screen for active
Kir3.2 channels subjected to random mutagenesis has identified residues
in the transmembrane segments that are crucial for controlling the
opening of Kir3.2 channels. The identification of structural elements
involved in potassium channel gating in these systems highlights
principles that may be important in the regulation of other types of
channels.
Functionalized xenon as a biosensor
Authors: Spence Megan M. , Rubin Seth M. , Dimitrov Ivan E. , Ruiz E. Janette , Wemmer David E. , Pines Alexander , Yao Shao Qin , Tian Feng , Schultz Peter G.
Published: July 2002

The detection of biological molecules and their interactions is a significant component of modern biomedical research. In current biosensor technologies, simultaneous detection is limited to a small number of analytes by the spectral overlap of their signals. We have developed an NMR-based xenon biosensor that capitalizes on the enhanced signal-to-noise, spectral simplicity, and chemical-shift sensitivity of laser-polarized xenon to detect specific biomolecules at the level of tens of nanomoles. We present results using xenon “functionalized” by a biotin-modified supramolecular cage to detect biotin–avidin binding. This biosensor methodology can be extended to a multiplexing assay for multiple analytes.
Resistance to Taxol in lung cancer cells associated with increased microtubule dynamics
Authors: Gonçalves A. , Braguer D. , Kamath K. , Martello L. , Briand C. , Horwitz S. , Wilson L. , Jordan M. A.
Published: July 2002

Microtubule dynamics are crucial for mitotic spindle assembly and
chromosome movement. Suppression of dynamics by Taxol appears
responsible for the drug's potent ability to inhibit mitosis and cell
proliferation. Although Taxol is an important chemotherapeutic agent,
development of resistance limits its efficacy. To examine the role of
microtubule dynamics in Taxol resistance, we measured the dynamic
instability of individual rhodamine-labeled microtubules in
Taxol-sensitive and -resistant living human cancer cells.
Taxol-resistant A549-T12 and -T24 cell lines were selected from a human
lung carcinoma cell line, A549. They are, respectively, 9- and 17-fold
resistant to Taxol and require low concentrations of Taxol for
proliferation. We found that microtubule dynamic instability was
significantly increased in the Taxol-resistant cells. For example, with
A549-T12 cells in the absence of added Taxol, microtubule dynamicity
increased 57% as compared with A549 cells. The length and rate of
shortening excursions increased 75 and 59%, respectively. These
parameters were further increased in A549-T24 cells, with overall
dynamicity increasing by 167% compared with parental cells. Thus, the
decreased Taxol-sensitivity of these cells can be explained by their
increased microtubule dynamics. When grown without Taxol, A549-T12
cells were blocked at the metaphase/anaphase transition and displayed
abnormal mitotic spindles with uncongressed chromosomes. In the
presence of 2–12 nM Taxol, the cells grew normally, suggesting that
mitotic block resulted from excessive microtubule dynamics. These
results indicate that microtubule dynamics play an important role in
Taxol resistance, and that both excessively rapid dynamics and
suppressed dynamics impair mitotic spindle function and inhibit
proliferation.
Human disease-causing
NOG
missense mutations: Effects on noggin secretion, dimer formation, and bone morphogenetic protein binding
Authors: Marcelino Jose , Sciortino Christopher M. , Romero Michael F. , Ulatowski Lynn M. , Ballock R. Tracy , Economides Aris N. , Eimon Peter M. , Harland Richard M. , Warman Matthew L.
Published: July 2002

Secreted noggin protein regulates bone morphogenetic protein
activity during development. In mice, a complete loss of noggin protein
leads to multiple malformations including joint fusion, whereas mice
heterozygous for
Nog
loss-of-function mutations are
normal. In humans, heterozygous
NOG
missense mutations
have been found in patients with two autosomal dominant disorders of
joint development, multiple synostosis syndrome (SYNS1) and a milder
disorder proximal symphalangism (SYM1). This study investigated the
effect of one SYNS1 and two SYM1 disease-causing missense mutations on
the structure and function of noggin. The SYNS1 mutation abolished, and
the SYM1 mutations reduced, the secretion of functional noggin dimers
in transiently transfected COS-7 cells. Coexpression of mutant noggin
with wild-type noggin, to resemble the heterozygous state, did not
interfere with wild-type noggin secretion. These data indicate that the
human disease-causing mutations are hypomorphic alleles that reduce
secretion of functional dimeric noggin. Therefore, we conclude that
noggin has both species-specific and joint-specific dosage-dependent
roles during joint formation. Surprisingly, in contrast to the COS-7
cell studies, the SYNS1 mutant was able to form dimers in
Xenopus
laevis
oocytes. This finding
indicates that there also exist species-specific differences in the
ability to process mutant noggin polypeptides.
Morphological properties of atmospheric aerosol aggregates
Authors: Xiong C. , Friedlander S. K.
Published: July 2002

Ultrafine particles (smaller than about 0.1 μm) are often emitted from combustion and other high-temperature processes in the form of fractal-like aggregates composed of solid nanoparticles. Results of a study of atmospheric aggregates are reported. Particles were collected on transmission electron microscope grids fitted on the last two stages of a single-jet eight-stage low-pressure impactor for periods of a few minutes. Photomicrographs of transmission electron microscope grids from the impactor stages were analyzed to obtain the fractal dimension (
D

f

) and prefactor (
A
) for aggregates.
D

f

increased from near 1 to above 2 as the number of primary particles making up the aggregates increased from 10 to 180. Total particle concentrations in size ranges roughly equivalent to the low-pressure impactor stages were measured with a mobility analyzer and condensation particle counter. In one set of measurements, the fraction of the particles present as aggregates was about 60% for particles with aerodynamic diameters between 50 and 75 nm and 34% for the range 75 to 120 nm. The total aggregate concentration in the 50- to 120-nm size range was about 400 ml
−1
. The primary particles that make up atmospheric aggregates are more polydisperse than soot aggregates generated from a single laboratory source, an ethane/oxygen flame. Most measurements were made in the Los Angeles area, where the aggregates may represent a signature for diesel emissions. Rural aggregate concentrations in the size range 50 to 120 nm were less than 1% of the concentrations at urban sites. The data will permit better estimates of atmospheric aggregate residence times, transport, and deposition in the lung, optical extinction, and heterogenous nucleation.
Zidovudine–didanosine coexposure potentiates DNA incorporation of zidovudine and mutagenesis in human cells
Authors: Meng Quanxin , Walker Dale M. , Olivero Ofelia A. , Shi Xiaochu , Antiochos Brendan B. , Poirier Miriam C. , Walker Vernon E.
Published: July 2002

Drug combinations that include nucleoside reverse transcriptase
inhibitors (NRTIs) are remarkably effective in preventing
maternal-viral transmission of HIV during pregnancy. However, there may
be potential long-term risks for children exposed
in
utero
. Examination of the genotoxic and mutagenic effects of
two NRTIs, zidovudine [AZT (3′-azido-3′-deoxythymidine)] and
didanosine [ddI (2′,3′-dideoxyinosine)], in cultured human
lymphoblastoid cells revealed multiplicative synergistic enhancement of
AZT-DNA incorporation and mutant frequency induction in response to the
combined drug exposure, as compared with single-drug exposures.
Dose-related increases in DNA incorporation of AZT (as measured by a
competitive RIA) and mutagenicity at the
HPRT
and
TK
loci (as assessed by cell-cloning assays) were
observed in cells exposed in culture to AZT, or equimolar combinations
of AZT + ddI, at exposure concentrations ranging from 3 to 30 times the
maximum plasma levels found in humans. Because mutagenesis is strongly
associated with tumor induction in experimental models, children
exposed transplacentally to combinations of NRTIs may be at risk for
cancer development later in life.
2′-Hydroxylation of nicotine by cytochrome P450 2A6 and human liver microsomes: Formation of a lung carcinogen precursor
Authors: Hecht Stephen S. , Hochalter J. Bradley , Villalta Peter W. , Murphy Sharon E.
Published: July 2002

Smokers or people undergoing nicotine replacement therapy
excrete approximately 10% of the nicotine dose as
4-oxo-4-(3-pyridyl)butanoic acid (keto acid) and
4-hydroxy-4-(3-pyridyl)butanoic acid (hydroxy acid). Previously, these
acids were thought to arise by secondary metabolism of the major
nicotine metabolite cotinine, but our data did not support this
mechanism. Therefore, we hypothesized that nicotine is metabolized by
2′-hydroxylation, which would ultimately yield keto acid and hydroxy
acid as urinary metabolites. This pathway had not been established
previously in mammalian systems and is potentially significant because
the product of nicotine 2′-hydroxylation,
4-(methylamino)-1-(3-pyridyl)-1-butanone (aminoketone), can be
converted to the potent tobacco-specific lung carcinogen,
4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone. Incubation of nicotine
with cytochrome P450 2A6 and cofactors did indeed produce aminoketone,
which was identified as its
N
-benzoyl derivative by
GC-MS. The rate was 11% of that of cotinine production. Incubation of
human liver microsomes with nicotine gave keto acid by using
aminoketone as an intermediate; keto acid was not formed from cotinine.
In 10 human liver samples, rates of formation of keto acid were 5.7%
of those of cotinine and production of these metabolites correlated.
These results provide definitive evidence for mammalian
2′-hydroxylation of nicotine and elucidate a pathway by which
endogenous formation of
4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone could occur in humans.
Role of C/EBP homologous protein (CHOP-10) in the programmed activation of CCAAT/enhancer-binding protein-β during adipogenesis
Authors: Tang Qi-Qun , Lane M. Daniel
Published: July 2002

Hormone induction of growth-arrested preadipocytes triggers mitotic
clonal expansion followed by expression of
CCAAT/enhancer-binding protein (C/EBP)α and
differentiation into adipocytes. The order of these events is critical
because C/EBPα is antimitotic and its expression prematurely
would block the mitotic clonal expansion required for differentiation.
C/EBPβ, a transcriptional activator of the C/EBPα
gene, is expressed early in the differentiation program, but lacks
DNA-binding activity and fails to localize to centromeres until
preadipocytes traverse the G
1
-S checkpoint of mitotic
clonal expansion. Evidence is presented that dominant-negative CHOP-10
expressed by growth-arrested preadipocytes transiently sequesters
C/EBPβ by heterodimerization. As preadipocytes reach S phase,
CHOP-10 is down-regulated, apparently releasing C/EBPβ from
inhibitory constraint and allowing transactivation of the
C/EBPα gene. In support of these findings, up-regulation of
CHOP-10 with the protease inhibitor
N
-acetyl-Leu-Leu-norleucinal prevents activation of
C/EBPβ, expression of C/EBPα, and adipogenesis.
Essential role of Glu-C66 for menaquinol oxidation indicates transmembrane electrochemical potential generation by
Wolinella succinogenes
fumarate reductase
Authors: Lancaster C. Roy D. , Groß Roland , Haas Alexander , Ritter Michaela , Mäntele Werner , Simon Jörg , Kröger Achim
Published: July 2002

Quinol:fumarate reductase (QFR) is a membrane protein complex that
couples the reduction of fumarate to succinate to the oxidation of
quinol to quinone, in a reaction opposite to that catalyzed by the
related enzyme succinate:quinone reductase (succinate dehydrogenase).
In the previously determined structure of QFR from
Wolinella
succinogenes
, the site of fumarate reduction in the
flavoprotein subunit A of the enzyme was identified, but the site of
menaquinol oxidation was not. In the crystal structure, the acidic
residue Glu-66 of the membrane spanning, diheme-containing subunit C
lines a cavity that could be occupied by the substrate menaquinol. Here
we describe that, after replacement of Glu-C66 with Gln by
site-directed mutagenesis, the resulting mutant is unable to grow on
fumarate and the purified enzyme lacks quinol oxidation activity. X-ray
crystal structure analysis of the Glu-C66 → Gln variant enzyme at
3.1-Å resolution rules out any major structural changes compared with
the wild-type enzyme. The oxidation-reduction potentials of the heme
groups are not significantly affected. We conclude that Glu-C66 is an
essential constituent of the menaquinol oxidation site. Because Glu-C66
is oriented toward a cavity leading to the periplasm, the release of
two protons on menaquinol oxidation is expected to occur to the
periplasm, whereas the uptake of two protons on fumarate reduction
occurs from the cytoplasm. Thus our results indicate that the reaction
catalyzed by
W. succinogenes
QFR generates a
transmembrane electrochemical potential.
Mechanism of indinavir-induced hyperbilirubinemia
Authors: Zucker Stephen D. , Qin Xiaofa , Rouster Susan D. , Yu Fei , Green Richard M. , Keshavan Pavitra , Feinberg Judith , Sherman Kenneth E.
Published: July 2002

Indinavir is a viral protease inhibitor used for the treatment of HIV infection. Unconjugated hyperbilirubinemia develops in up to 25% of patients receiving indinavir, prompting drug discontinuation and further clinical evaluation in some instances. We postulated that this side-effect is due to indinavir-mediated impairment of bilirubin UDP-glucuronosyltransferase (UGT) activity and would be most pronounced in individuals with reduced hepatic enzyme levels, as occurs in ≈10% of the population manifesting Gilbert's syndrome. This hypothesis was tested
in vitro
, in the Gunn rat model of UGT deficiency, and in HIV-infected patients with and without the Gilbert's polymorphism. Indinavir was found to competitively inhibit UGT enzymatic activity (
K
I
= 183 μM) while concomitantly inducing hepatic bilirubin UGT mRNA and protein expression. Although oral indinavir increased plasma bilirubin levels in wild-type and heterozygous Gunn rats, the mean rise was significantly greater in the latter group of animals. Similarly, serum bilirubin increased by a mean of 0.34 mg/dl in indinavir-treated HIV patients lacking the Gilbert's polymorphism versus 1.45 mg/dl in those who were either heterozygous or homozygous for the mutant allele. Whereas saquinavir also competitively inhibits UGT activity, this drug has not been associated with hyperbilirubinemia, most likely because of the higher
K
I
(360 μM) and substantially lower therapeutic levels as compared with indinavir. Taken together, these findings indicate that elevations in serum-unconjugated bilirubin associated with indinavir treatment result from direct inhibition of bilirubin-conjugating activity.
Distinction between signaling mechanisms in lipid rafts vs. caveolae
Authors: Sowa Grzegorz , Pypaert Marc , Sessa William C.
Published: July 2002

The relative importance of lipid rafts vs. specialized rafts termed caveolae to influence signal transduction is not known. Here we show that in cells lacking caveolae, the dually acylated protein, endothelial nitric oxide synthase (eNOS), localizes to cholesterol-rich lipid raft domains of the plasma membrane. In these cells, expression of caveolin-1 (cav-1) stimulates caveolae biogenesis, promotes the interaction of cav-1 with eNOS, and the inhibition of NO release from cells. Interestingly, in cells where cav-1 does not drive caveolae assembly, despite equal levels of cav-1 and eNOS and localization of both proteins to raft domains of the plasmalemma, the physical interaction of eNOS with cav-1 is dramatically less resulting in less inhibition of NO release. Thus, cav-1 concentrated in caveolae, not in rafts, is in closer proximity to eNOS and is necessary for negative regulation of eNOS function, thereby providing the first clear example of spatial regulation of signaling in this organelle that is distinct from raft domains.
Rapid formation of the stable tyrosyl radical in photosystem II
Authors: Faller Peter , Debus Richard J. , Brettel Klaus , Sugiura Miwa , Rutherford A. William , Boussac Alain
Published: July 2002

Two symmetrically positioned redox active tyrosine residues are present in the photosystem II (PSII) reaction center. One of them, TyrZ, is oxidized in the ns–μs time scale by P680+and reduced rapidly (μs to ms) by electrons from the Mn complex. The other one, TyrD, is stable in its oxidized form and seems to play no direct role in enzyme function. Here, we have studied electron donation from these tyrosines to the chlorophyll cation (P680+) in Mn-depleted PSII from plants and cyanobacteria. In particular, a mutant lacking TyrZ was used to investigate electron donation from TyrD. By using EPR and time-resolved absorption spectroscopy, we show that reduced TyrD is capable of donating an electron to P680+with t1/2≈ 190 ns at pH 8.5 in approximately half of the centers. This rate is ≈105times faster than was previously thought and similar to the TyrZ donation rate in Mn-depleted wild-type PSII (pH 8.5). Some earlier arguments put forward to rationalize the supposedly slow electron donation from TyrD (compared with that from TyrZ) can be reassessed. At pH 6.5, TyrZ (t1/2= 2–10 μs) donates much faster to P680+than does TyrD (t1/2> 150 μs). These different rates may reflect the different fates of the proton released from the respective tyrosines upon oxidation. The rapid rate of electron donation from TyrD requires at least partial localization of P680+on the chlorophyll (PD2) that is located on the D2 side of the reaction center.
Dynamic mechanisms of the membrane water channel aquaporin-1 (AQP1)
Authors: Kong Yifei , Ma Jianpeng
Published: July 2002

Molecular-dynamics simulations were performed on the
structures of the water channel aquaporin-1. The results provide
an atomistic description of the interactions involved in the water
permeation. Two major curvilinear pathways were identified. The
simulations confirmed that the water selectivity is due primarily to
the size-exclusion effect; i.e., maximally, one water molecule is
allowed to pass through the narrow constriction in the aqueous pathway.
Most importantly, in contrast to previous proposals, the
hydrogen-bonding interactions of water molecules with the polar side
chains of Asn-76 and Asn-192 on the strictly conserved Asn-Pro-Ala
sequence motifs were found to be essential for maintaining the
connectivity of water flow in the narrow constriction region. When
Asn-76 and Asn-192 were replaced with near-isosteric hydrophobic
residues in the simulation, the aqueous pathways were broken
completely. Additionally, the size of the narrow constriction
fluctuates significantly during the simulation, which frequently breaks
the flow of water and, thus, breaks the single-file water network
necessary for proton translocation. Moreover, mutations based on the
simulation also have been suggested for further experimental
investigation of the water-permeation mechanism of aquaporin-1.
Lipopolysaccharide-induced leptin release is neurally controlled
Authors: Mastronardi C. A. , Yu W. H. , Srivastava V. K. , Dees W. L. , McCann S. M.
Published: July 2002

Our hypothesis is that leptin release is controlled
neurohormonally. Conscious, male rats bearing indwelling,
external, jugular catheters were injected with the test drug or 0.9%
NaCl (saline), and blood samples were drawn thereafter to measure
plasma leptin. Anesthesia decreased plasma leptin concentrations within
10 min to a minimum at 120 min, followed by a rebound at 360 min.
Administration (i.v.) of lipopolysaccharide (LPS) increased plasma
leptin to almost twice baseline by 120 min, and it remained on a
plateau for 360 min, accompanied by increased adipocyte leptin mRNA.
Anesthesia largely blunted the LPS-induced leptin release at 120 min.
Isoproterenol (β-adrenergic agonist) failed to alter plasma leptin
but reduced LPS-induced leptin release significantly. Propranolol
(β-receptor antagonist) produced a significant increase in plasma
leptin but had no effect on the response to LPS. Phentolamine
(α-adrenergic receptor blocker) not only increased plasma leptin
(
P
< 0.001), but also augmented the LPS-induced
increase (
P
< 0.001). α-Bromoergocryptine
(dopaminergic-2 receptor agonist) decreased plasma leptin
(
P
< 0.01) and blunted the LPS-induced rise in
plasma leptin release (
P
< 0.001). We conclude
that leptin is at least in part controlled neurally because anesthesia
decreased plasma leptin and blocked its response to LPS. The findings
that phentolamine and propranolol increased plasma leptin
concentrations suggest that leptin release is inhibited by the
sympathetic nervous system mediated principally by α-adrenergic
receptors because phentolamine, but not propranolol, augmented the
response to LPS. Because α-bromoergocryptine decreased basal and
LPS-induced leptin release, dopaminergic neurons may inhibit basal and
LPS-induced leptin release by suppression of release of prolactin from
the adenohypophysis.
Peripheral myelin protein 22 is a constituent of intercellular junctions in epithelia
Authors: Notterpek Lucia , Roux Kyle J. , Amici Stephanie A. , Yazdanpour Amy , Rahner Christoph , Fletcher Brad S.
Published: July 2002

Alterations in peripheral myelin protein 22 (PMP22) gene expression
are associated with a host of heritable demyelinating peripheral
neuropathies, yet the function of the protein remains unknown.
PMP22 expression is highest in myelinating Schwann cells of peripheral
nerves; however, significant levels of PMP22 mRNAs can be detected in a
variety of non-neural tissue, including epithelia. To date, PMP22
protein expression and localization in non-neural tissues have not been
studied in detail. In adult rat liver and intestine, and cultured
epithelial cells, we detected PMP22-like immunoreactivity associated
with markers of the tight junctional complex, including zonula
occludens 1 (ZO-1) and occludin. Upon disruption of intercellular
contacts, PMP22 was internalized into vesicles that were immunoreactive
for both anti-occludin and anti-PMP22 antibodies. Nonionic detergent
extraction of cultured epithelial cells did not solubilize PMP22, as
the majority of the protein remained in the detergent insoluble
fraction, as did ZO-1 and occludin. We also observed the targeting of
exogenous myc-tagged PMP22 to apical cell junctions in polarized
epithelia and to anti-ZO-1 antibody immunoreactive cell contacts of L
fibroblasts. These studies support a role for PMP22 at intercellular
junctions of epithelia and may indicate a similar function in
myelinating Schwann cells. Furthermore, our findings could provide an
explanation for certain phenotypes of PMP22 neuropathy mice that cannot
be accounted for by dysmyelination.
Oligodeoxynucleotides inhibit retinal neovascularization in a murine model of proliferative retinopathy.
Authors: Robinson G S , Pierce E A , Rook S L , Foley E , Webb R , Smith L E
Published: July 2002

Diseases characterized by retinal neovascularization are among the principal causes of visual loss worldwide. The hypoxia-stimulated expression of vascular endothelial growth factor (VEGF) has been implicated in the proliferation of new blood vessels. We have investigated the use of antisense phosphorothioate oligodeoxynucleotides against murine VEGF to inhibit retinal neovascularization and VEGF synthesis in a murine model of proliferative retinopathy. Intravitreal injections of two different antisense phosphorothioate oligodeoxynucleotides prior to the onset of proliferative retinopathy reduced new blood vessel growth a mean of 25 and 31% compared with controls. This inhibition was dependent on the concentration of antisense phosphorothioate oligodeoxynucleotides and resulted in a 40-66% reduction in the level of VEGF protein, as determined by Western blot analysis. Control (sense, nonspecific) phosphorothioate oligodeoxynucleotides did not cause a significant reduction in retinal neovascularization or VEGF protein levels. These data further establish a fundamental role for VEGF expression in ischemia-induced proliferative retinopathies and a potential therapeutic use for antisense phosphorothioate oligodeoxynucleotides.
Functional expression of the Schizosaccharomyces pombe Na+/H+ antiporter gene, sod2, in Saccharomyces cerevisiae.
Authors: Hahnenberger K M , Jia Z , Young P G
Published: July 2002

In the fission yeast, Schizosaccharomyces pombe, tolerance to high sodium and lithium concentrations requires the functioning of the sod2, Na+/H+ antiporter. We have directly measured the activity of this antiporter and demonstrated reconstitution of the activity in gene deletion strains. In addition, we have shown that it can be transferred to, and its antiporter activity detected in, the budding yeast, Saccharomyces cerevisiae, where it also confers sodium and lithium tolerance. Proton flux through the S. pombe Na+/H+ antiporter was directly measured using microphysiometry. The direction of transmembrane proton flux mediated by this antiporter was reversible, with protons being imported or exported in response to the external concentration of sodium. This bidirectional activity was also detected in S. cerevisiae strains expressing sod2 and expression of this gene complemented the sodium and lithium sensitivity resulting from inactivation of the ENA1/PMR2 encoded Na+-exporting ATPases. This suggests that antiporters or sodium pumps can be utilized interchangeably by S. cerevisiae to regulate internal sodium concentration. Potent inhibitors of mammalian Na+/H+ exchangers were found to have no effect on sod2 activity. The proton flux mediated by sod2 was also found to be unaffected by perturbation of membrane potential or the plasma membrane proton gradient.
Glucocorticoid and progestin receptors are differently involved in the cooperation with a structural element of the mouse mammary tumor virus promoter.
Authors: Le Ricousse S , Gouilleux F , Fortin D , Joulin V , Richard-Foy H
Published: July 2002

We have previously characterized a regulatory element located between -294 and -200 within the mouse mammary tumor virus (MMTV) long terminal repeat (LTR). This element termed AA element cooperates with the glucocorticoid response elements (GREs) for glucocorticoid activation. Here we show that in a MMTV LTR wild type context, the deletion of this element significantly reduces both glucocorticoid and progestin activation of the promoter. Deletion of the two most distal GREs forces the glucocorticoid receptor (GR) and the progestin receptor (PR) to bind the same response elements and results in a dramatic decrease in the inducibility of the MMTV promoter by the two hormones. The simultaneous deletion of the two distal GREs and of the AA element abolishes completely the glucocorticoid-induced activation of the promoter. In contrast it restores a significant level of progestin-induced activation. This different effect of the double deletion on glucocorticoid- and progestin-induced MMTV promoter activation is not cell specific because it is also observed, and is even stronger, when either GR or PR is expressed in the same cell line (NIH 3T3). This is the first description of a mutated MMTV promoter that, although retaining GREs, is activated by progestins and not by glucocorticoids. This suggests a different functional cooperation between protein(s) interacting with the AA element and GR or PR. Cotransfections with constructs containing wild-type or mutated MMTV LTR with either PR lacking its C-terminal domain or GR/PR chimeras in which the N-terminal domains have been exchanged demonstrate that the N-terminal domains of the receptors specify the different behavior of GR and PR regarding the AA element.