bluuebunny/crossref_metadata_2025 huggingface dataset:
Photic and circadian expression of luciferase in
mPeriod1
-
luc
transgenic mice
in
vivo
Authors: Wilsbacher Lisa D. , Yamazaki Shin , Herzog Erik D. , Song Eun-Joo , Radcliffe Laurel A. , Abe Michikazu , Block Gene , Spitznagel Edward , Menaker Michael , Takahashi Joseph S.
Published: July 2002

A conserved transcription-translation negative feedback loop
forms the molecular basis of the circadian oscillator in animals.
Molecular interactions within this loop have been relatively well
characterized
in vitro
and in cell culture; however,
in vivo
approaches are required to assess the functional
significance of these interactions. Here, regulation of circadian gene
expression was studied
in vivo
by using transgenic
reporter mouse lines in which 6.75 kb of the mouse
Period1
(
mPer1
) promoter drives
luciferase
(
luc
) expression. Six
mPer1
-
luc
transgenic lines were created,
and all lines express a daily rhythm of
luc
mRNA in the
suprachiasmatic nuclei (SCN). Each
mPer1
-
luc
line also sustains a long-term
circadian rhythm of luminescence in SCN slice culture. A 6-h light
pulse administered during the early subjective night rapidly induces
luc
mRNA expression in the SCN; however, high
luc
mRNA levels are sustained, whereas
endogenous
mPer1
mRNA levels return to
baseline, suggesting that posttranscriptional events mediate the
down-regulation of
mPer1
after exposure to light. This
approach demonstrates that the 6.75-kb
mPer1
promoter
fragment is sufficient to confer both circadian and photic regulation
in vivo
and reveals a potential posttranscriptional
regulatory mechanism within the mammalian circadian oscillator.
Mechanism of action and NAD
+
-binding mode revealed by the crystal structure of
l
-histidinol dehydrogenase
Authors: Barbosa João A. R. G. , Sivaraman J. , Li Yunge , Larocque Robert , Matte Allan , Schrag Joseph D. , Cygler Miroslaw
Published: July 2002

The histidine biosynthetic pathway is an ancient one found in bacteria, archaebacteria, fungi, and plants that converts 5-phosphoribosyl 1-pyrophosphate to
l
-histidine in 10 enzymatic reactions. This pathway provided a paradigm for the operon, transcriptional regulation of gene expression, and feedback inhibition of a pathway.
l
-histidinol dehydrogenase (HisD, EC
1.1.1.23
) catalyzes the last two steps in the biosynthesis of
l
-histidine: sequential NAD-dependent oxidations of
l
-histidinol to
l
-histidinaldehyde and then to
l
-histidine. HisD functions as a homodimer and requires the presence of one Zn
2+
cation per monomer. We have determined the three-dimensional structure of
Escherichia coli
HisD in the apo state as well as complexes with substrate, Zn
2+
, and NAD
+
(best resolution is 1.7 Å). Each monomer is made of four domains, whereas the intertwined dimer possibly results from domain swapping. Two domains display a very similar incomplete Rossmann fold that suggests an ancient event of gene duplication. Residues from both monomers form the active site. Zn
2+
plays a crucial role in substrate binding but is not directly involved in catalysis. The active site residue His-327 participates in acid-base catalysis, whereas Glu-326 activates a water molecule. NAD
+
binds weakly to one of the Rossmann fold domains in a manner different from that previously observed for other proteins having a Rossmann fold.
Calcium signaling: A tale for all seasons
Authors: Carafoli Ernesto
Published: July 2002

An experiment performed in London nearly 120 years ago, which by today's standards would be considered unacceptably sloppy, marked the beginning of the calcium (Ca
2+
) signaling saga. Sidney Ringer [Ringer, S. (1883)
J. Physiol.
4, 29–43] was studying the contraction of isolated rat hearts. In earlier experiments, Ringer had suspended them in a saline medium for which he admitted to having used London tap water, which is hard: The hearts contracted beautifully. When he proceeded to replace the tap water with distilled water, he made a startling finding: The beating of the hearts became progressively weaker, and stopped altogether after about 20 min. To maintain contraction, he found it necessary to add Ca
2+
salts to the suspension medium. Thus, Ringer had serendipitously discovered that Ca
2+
, hitherto exclusively considered as a structural element, was active in a tissue that has nothing to do with bone or teeth, and performed there a completely novel function: It carried the signal that initiated heart contraction. It was a landmark observation, which should have immediately aroused wide interest. Unexpectedly, however, for decades it attracted no particular attention. Occasionally, farsighted pioneers argued forcefully for a messenger role of Ca
2+
, offering compelling experimental evidence. Among them, one could quote L. V. Heilbrunn [Heilbrunn, L. V. (1940)
Physiol. Zool.
13, 88–94], who contracted frog muscle fibers by applying Ca
2+
salts to their cut ends, but not to their surfaces. Heilbrunn correctly concluded that Ca
2+
had diffused from the cut ends to the internal contractile elements to elicit their contraction. One could also quote K. Bailey [Bailey, K. (1942)
Biochem. J.
36, 121–139], who showed that the ATPase activity of myosin was strongly activated by Ca
2+
(but not by Mg
2+
), and concluded that the liberation of Ca
2+
in the neighborhood of the myosin controlled muscle contraction. Clearly, enough evidence was there, but only a handful of people had the vision to see it and to foresee its far-reaching implications. Perhaps no better example of clairvoyance can be offered than the quip by O. Loewy in 1959: “Ja Kalzium, das ist alles!”
Representation of actions in rats: The role of cerebellum in learning spatial performances by observation
Authors: Leggio Maria G. , Molinari Marco , Neri Paola , Graziano Alessandro , Mandolesi Laura , Petrosini Laura
Published: July 2002

Experimental evidence demonstrates that cerebellar networks are involved in spatial learning, controlling the acquisition of exploration strategies without blocking motor execution of the task. Action learning by observation has been considered somehow related to motor physiology, because it provides a way of learning performances that is almost as effective as the actual execution of actions. Neuroimaging studies demonstrate that observation of movements performed by others, imagination of actions, and actual execution of motor performances share common neural substrates and that the cerebellum is among these shared areas. The present paper analyzes the effects of observation in learning a spatial task, focusing on the cerebellar role in learning a spatial ability through observation. We allowed normal rats to observe 200 Morris water maze trials performed by companion rats. After this observation training, “observer” rats underwent a hemicerebellectomy and then were tested in the Morris water maze. In spite of the cerebellar lesion, they displayed no spatial defects, exhibiting exploration abilities comparable to controls. When the cerebellar lesion preceded observation training, a complete lack of spatial observational learning was observed. Thus, as demonstrated already for the acquisition of spatial procedures through actual execution, cerebellar circuits appear to play a key role in the acquisition of spatial procedures also through observation. In conclusion, the present results provide strong support for a common neural basis in the observation of actions that are to be reproduced as well as in the actual production of the same actions.
Inhibition of caspase 1 reduces human myocardial ischemic dysfunction via inhibition of IL-18 and IL-1β
Authors: Pomerantz Benjamin J. , Reznikov Leonid L. , Harken Alden H. , Dinarello Charles A.
Published: July 2002

The proinflammatory cytokine IL-18 was investigated for its role in
human myocardial function. An ischemia/reperfusion (I/R) model of
suprafused human atrial myocardium was used to assess myocardial
contractile force. Addition of IL-18 binding protein (IL-18BP), the
constitutive inhibitor of IL-18 activity, to the perifusate during and
after I/R resulted in improved contractile function after I/R from
35% of control to 76% with IL-18BP. IL-18BP treatment also preserved
intracellular tissue creatine kinase levels (by 420%). Steady-state
mRNA levels for IL-18 were elevated after I/R, and the concentration
of IL-18 in myocardial homogenates was increased (control, 5.8 pg/mg
vs. I/R, 26 pg/mg;
P
< 0.01). Active IL-18
requires cleavage of its precursor form by the IL-1β-converting
enzyme (caspase 1); inhibition of caspase 1 also attenuated the
depression in contractile force after I/R (from 35% of control to
75.8% in treated atrial muscle;
P
< 0.01).
Because caspase 1 also cleaves the precursor IL-1β, IL-1 receptor
blockade was accomplished by using the IL-1 receptor antagonist. IL-1
receptor antagonist added to the perifusate also resulted in a
reduction of ischemia-induced contractile dysfunction. These studies
demonstrate that endogenous IL-18 and IL-1β play a
significant role in I/R-induced human myocardial injury and that
inhibition of caspase 1 reduces the processing of
endogenous precursors of IL-18 and IL-1β and
thereby prevents ischemia-induced myocardial dysfunction.
Analysis of the ultraviolet B response in primary human keratinocytes using oligonucleotide microarrays
Authors: Sesto Angela , Navarro Manuel , Burslem Frank , Jorcano José L.
Published: July 2002

UV radiation is the most important environmental skin aggressor, causing cancer and other problems. This paper reports the use of oligonucleotide microarray technology to determine changes in gene expression in human keratinocytes after UVB treatment. Examination of the effects of different doses at different times after irradiation gave a global picture of the keratinocyte response to this type of insult. Five hundred thirty-nine regulated transcripts were found and organized into nine different clusters depending on behavior patterns. Classification of these genes into 23 functional categories revealed that several biological processes are globally affected by UVB. In addition to confirming a majority up-regulation of the transcripts related to the UV-specific inflammatory and stress responses, significant increases were seen in the expression of genes involved in basal transcription, splicing, and translation as well as in the proteasome-mediated degradation category. On the other hand, those transcripts belonging to the metabolism and adhesion categories were strongly downregulated. These results demonstrate the complexity of the transcriptional profile of the UVB response, describe several cellular processes previously not known to be affected by UV irradiation, and serve as a basis for the global characterization of UV-regulated genes and pathways.
Herpesviruses use bidirectional fast-axonal transport to spread in sensory neurons
Authors: Smith Gregory A. , Gross Steven P. , Enquist Lynn W.
Published: July 2002

Alpha herpesviruses infect the vertebrate nervous system resulting in either mild recurrent lesions in mucosal epithelia or fatal encephalitis. Movement of virions within the nervous system is a critical factor in the outcome of infection; however, the dynamics of individual virion transport have never been assessed. Here we visualized and tracked individual viral capsids as they moved in axons away from infected neuronal cell bodies in culture. The observed movement was compatible with fast axonal flow mediated by multiple microtubule motors. Capsids accumulated at axon terminals, suggesting that spread from infected neurons required cell contact.
Initiation of clement surface conditions on the earliest Earth
Authors: Sleep N. H. , Zahnle K. , Neuhoff P. S.
Published: July 2002

In the beginning the surface of the Earth was extremely hot, because the Earth as we know it is the product of a collision between two planets, a collision that also created the Moon. Most of the heat within the very young Earth was lost quickly to space while the surface was still quite hot. As it cooled, the Earth's surface passed monotonically through every temperature regime between silicate vapor to liquid water and perhaps even to ice, eventually reaching an equilibrium with sunlight. Inevitably the surface passed through a time when the temperature was around 100°C at which modern thermophile organisms live. How long this warm epoch lasted depends on how long a thick greenhouse atmosphere can be maintained by heat flow from the Earth's interior, either directly as a supplement to insolation, or indirectly through its influence on the nascent carbonate cycle. In both cases, the duration of the warm epoch would have been controlled by processes within the Earth's interior where buffering by surface conditions played little part. A potentially evolutionarily significant warm period of between 105and 107years seems likely, which nonetheless was brief compared to the vast expanse of geological time.
Developmental specificity of recruitment of TBP to the TATA box of the human γ-globin gene
Authors: Duan Zhi-Jun , Fang Xiangdong , Rohde Alex , Han Hemei , Stamatoyannopoulos George , Li Qiliang
Published: July 2002

It is unclear whether the core promoter is involved in developmental regulation. To address this question, we mutated the TATA box of the human γ-globin gene, produced transgenic mice, and examined the effect of the mutation during the course of mouse development. In our test system, the γ-globin gene is expressed at similar levels in the embryonic and adult erythroid cells. The TATA box mutation dramatically reduced expression of the γ-globin gene in the adult but not in embryonic erythroid cells. In addition, the disruption of the γ TATA box significantly reduced the recruitment of TATA box-binding protein (TBP) in the adult cells, but not in embryonic cells, suggesting that the recruitment of TBP to the γ gene promoter is developmentally specific. Similarly, the recruitment of transcription factor II B and RNA polymerase II to the γ promoter was affected in the adult but not in embryonic cells. The distinct effects of the TATA mutation in the embryonic and adult developmental stages suggest that the basal transcription apparatus can be recruited to a core promoter in a developmental stage-dependent manner. The TATA mutation resulted in a shift of transcription initiation site 6 bp or longer upstream to the cap site both in the embryonic and adult erythrocytes. We conclude that the TATA box determines the initiation site but not the efficiency of transcription of the γ-globin gene.
Geometric requirements and examples of important structures in the assembly of square building blocks
Authors: Eddaoudi Mohamed , Kim Jaheon , Vodak David , Sudik Andrea , Wachter Joseph , O'Keeffe Michael , Yaghi Omar M.
Published: July 2002

The basic structures for linking squares into polyhedra and networks (reticulation) are enumerated, and corresponding examples are described in which crystals were synthesized by linking paddle wheel (square) units into metal–organic frameworks (MOFs)—named MOF-102 to MOF-112.
Networks of interneurons with fast and slow γ-aminobutyric acid type A (GABA
A
) kinetics provide substrate for mixed gamma-theta rhythm
Authors: White John A. , Banks Matthew I. , Pearce Robert A. , Kopell Nancy J.
Published: July 2002

During active exploration, hippocampal neurons exhibit nested rhythmic activity at theta (≈8 Hz) and gamma (≈40 Hz) frequencies. Gamma rhythms may be generated locally by interactions within a class of interneurons mediating fast GABA
A
(GABA
A,fast
) inhibitory postsynaptic currents (IPSCs), whereas theta rhythms traditionally are thought to be imposed extrinsically. However, the hippocampus contains slow biophysical mechanisms that may contribute to the theta rhythm, either as a resonance activated by extrinsic input or as a purely local phenomenon. For example, region CA1 of the hippocampus contains a slower class of GABA
A
(GABA
A,slow
) synapses, believed to be generated by a distinct group of interneurons. Recent evidence indicates that these GABA
A,slow
interneurons project to the GABA
A,fast
interneurons that contribute to hippocampal gamma rhythms. Here, we use biophysically based simulations to explore the possible ramifications of interneuronal circuits containing separate classes of GABA
A,fast
and GABA
A,slow
interneurons. Simulated interneuronal networks with fast and slow synaptic kinetics can generate mixed theta-gamma rhythmicity under restricted conditions, including strong connections among each population, weaker connections between the two populations, and homogeneity of cellular properties and drive. Under a broader range of conditions, including heterogeneity, the networks can amplify and resynchronize phasic responses to weak phase-dispersed external drive at theta frequencies to either GABA
A,slow
or GABA
A,fast
cells. GABA
A,slow
synapses are necessary for this process of amplification and resynchronization.
Kinetics and thermodynamics of T cell receptor– autoantigen interactions in murine experimental autoimmune encephalomyelitis
Authors: Garcia K. Christopher , Radu Caius G. , Ho Joseph , Ober Raimund J. , Ward E. Sally
Published: July 2002

In the current study, cellular and molecular approaches have been used to analyze the biophysical nature of T cell receptor (TCR)–peptide MHC (pMHC) interactions for two autoreactive TCRs. These two TCRs recognize the N-terminal epitope of myelin basic protein (MBP1–11) bound to the MHC class II protein, I-Au, and are associated with murine experimental autoimmune encephalomyelitis. Mice transgenic for the TCRs have been generated and characterized in other laboratories. These analyses indicate that the mice either develop encephalomyelitis spontaneously (172.10 TCR) or only if immunized with autoantigen in adjuvant (1934.4 TCR). Here, we show that the 172.10 TCR binds MBP1–11:I-Auwith a 4–5-fold higher affinity than the 1934.4 TCR. Consistent with the higher affinity, 172.10 T hybridoma cells are significantly more responsive to autoantigen than 1934.4 cells. The interaction of the 172.10 TCR with cognate ligand is more entropically unfavorable than that of the 1934.4 TCR, indicating that the 172.10 TCR undergoes greater conformational rearrangements upon ligand binding. The studies therefore suggest a correlation between the strength and plasticity of a TCR–pMHC interaction and the frequency of spontaneous disease in the corresponding TCR transgenic mice. The comparative analysis of these two TCRs has implications for understanding autoreactive T cell recognition and activation.
Presenilin-1 mutations of leucine 166 equally affect the generation of the Notch and APP intracellular domains independent of their effect on Aβ
42
production
Authors: Moehlmann Tobias , Winkler Edith , Xia Xuefeng , Edbauer Dieter , Murrell Jill , Capell Anja , Kaether Christoph , Zheng Hui , Ghetti Bernardino , Haass Christian , Steiner Harald
Published: July 2002

The Alzheimer's disease (AD)-associated presenilin (PS) proteins are required for the γ-secretase cleavages of the β-amyloid precursor protein and the site 3 (S3) protease cleavage of Notch. These intramembrane cleavages release amyloid-β peptide (Aβ), including the pathogenic 42-aa variant (Aβ
42
), as well as the β-amyloid precursor protein and the Notch intracellular domains (AICD, NICD). Whereas Aβ is generated by endoproteolysis in the middle of the transmembrane domain, AICD and NICD are generated by cleavages at analogous positions close to the cytoplasmic border of the transmembrane domain. Numerous mutations causing familial AD (FAD) that all cause increased production of Aβ
42
have been found in the PS1 gene. Here we have investigated the previously uncharacterized, very aggressive FAD mutation L166P that causes onset of AD in adolescence. Strikingly, the PS1 L166P mutation not only induces an exceptionally high increase of Aβ
42
production but also impairs NICD production and Notch signaling, as well as AICD generation. Thus, FAD-associated PS mutants cannot only affect the generation of NICD, but also that of AICD. Moreover, further analysis with artificial L166 mutants revealed that the γ-secretase cleavage at position 40/42 and the S3-like γ-secretase cleavage at position 49 of the Aβ domain are both differentially affected by PS1 L166 mutants. Finally, we show that PS1 L166 mutants affect the generation of NICD and AICD in a similar manner, supporting the concept that S3 protease and S3-like γ-secretase cleavages are mediated by identical proteolytic activities.
An atmospheric
p
CO
2
reconstruction across the Cretaceous-Tertiary boundary from leaf megafossils
Authors: Beerling D. J. , Lomax B. H. , Royer D. L. , Upchurch G. R. , Kump L. R.
Published: July 2002

The end-Cretaceous mass extinctions, 65 million years ago, profoundly influenced the course of biotic evolution. These extinctions coincided with a major extraterrestrial impact event and massive volcanism in India. Determining the relative importance of each event as a driver of environmental and biotic change across the Cretaceous-Tertiary boundary (KTB) crucially depends on constraining the mass of CO
2
injected into the atmospheric carbon reservoir. Using the inverse relationship between atmospheric CO
2
and the stomatal index of land plant leaves, we reconstruct Late Cretaceous-Early Tertiary atmospheric CO
2
concentration (
p
CO
2
) levels with special emphasis on providing a
p
CO
2
estimate directly above the KTB. Our record shows stable Late Cretaceous/Early Tertiary background
p
CO
2
levels of 350–500 ppm by volume, but with a marked increase to at least 2,300 ppm by volume within 10,000 years of the KTB. Numerical simulations with a global biogeochemical carbon cycle model indicate that CO
2
outgassing during the eruption of the Deccan Trap basalts fails to fully account for the inferred
p
CO
2
increase. Instead, we calculate that the postboundary
p
CO
2
rise is most consistent with the instantaneous transfer of ≈4,600 Gt C from the lithic to the atmospheric reservoir by a large extraterrestrial bolide impact. A resultant climatic forcing of +12 W⋅m
−2
would have been sufficient to warm the Earth's surface by ≈7.5°C, in the absence of counter forcing by sulfate aerosols. This finding reinforces previous evidence for major climatic warming after the KTB impact and implies that severe and abrupt global warming during the earliest Paleocene was an important factor in biotic extinction at the KTB.
Mutations in
Mlph
, encoding a member of the Rab effector family, cause the melanosome transport defects observed in
leaden
mice
Authors: Matesic Lydia E. , Yip Richard , Reuss Andreé E. , Swing Deborah A. , O'Sullivan T. Norene , Fletcher Colin F. , Copeland Neal G. , Jenkins Nancy A.
Published: July 2002

The
d, ash,
and
ln
coat color mutations provide a unique model system for the study of vesicle transport in mammals. All three mutant loci encode genes that are required for the polarized transport of melanosomes, the specialized, pigment-containing organelles of melanocytes, to the neighboring keratinocytes and eventually into coat hairs. Genetic studies suggest that these genes function in the same or overlapping pathways and are supported by biochemical studies showing that
d
encodes an actin-based melanosome transport motor, MyoVa, whereas
ash
encodes Rab27a, a protein that localizes to the melanosome and is postulated to serve as the MyoVa receptor. Here we show that
ln
encodes melanophilin (Mlph), a previously undescribed protein with homology to Rab effectors such as granuphilin, Slp3-a, and rabphilin-3A. Like all of these effectors, Mlph possesses two Zn
2+
-binding CX
2
CX
13,14
CX
2
C motifs and a short aromatic-rich amino acid region that is critical for Rab binding. However, Mlph does not contain the two Ca
2+
-binding C
2
domains found in these and other proteins involved in vesicle transport, suggesting that it represents a previously unrecognized class of Rab effectors. Collectively, our data show that Mlph is a critical component of the melanosome transport machinery and suggest that Mlph might function as part of a transport complex with Rab27a and MyoVa.
Prudent
Protomognathus
and despotic
Leptothorax duloticus
: Differential costs of ant slavery
Authors: Hare James F. , Alloway Thomas M.
Published: July 2002

The concept of ant slavery rests on the untested assumption that slave-making ants impose fitness costs on colonies of the species they raid. We tested that assumption by comparing the summertime seasonal productivity of
Leptothorax
spp. colonies in field exclosures without slavemakers, with a colony of the obligatory slave-making ant
Protomognathus americanus
, or with a colony of the obligatory slavemaker
Leptothorax duloticus
.
Leptothorax longispinosus
colonies placed in exclosures with
P. americanus
colonies did not differ significantly in any demographic attribute from colonies in exclosures without slavemakers. By contrast,
Leptothorax curvispinosus
colonies exposed to
L. duloticus
experienced significant reductions in dealate queens, workers, and larvae relative to control colonies exclosed without slavemakers. The pronounced difference in the impact of these slavemakers on their host-species populations correlates with differences in the behavior of the slavemakers observed in the laboratory and likely explains why
P. americanus
is more abundant than
L. duloticus
in nature. It seems that more advanced social parasites, like anatomical parasites, evolve to minimize their impact on their hosts, and thus can be regarded as “prudent social parasites.”
Bestrophin, the product of the Best vitelliform macular dystrophy gene (
VMD2
), localizes to the basolateral plasma membrane of the retinal pigment epithelium
Authors: Marmorstein Alan D. , Marmorstein Lihua Y. , Rayborn Mary , Wang Xinxing , Hollyfield Joe G. , Petrukhin Konstantin
Published: July 2002

Best vitelliform macular dystrophy is a dominantly inherited, early
onset, macular degenerative disease that exhibits some histopathologic
similarities to age-related macular degeneration. Although the
vitelliform lesion is common in the fundus of individuals with Best
disease, diagnosis is based on a reduced ratio of the light peak to
dark trough in the electrooculogram. Recently, the
VMD2
gene on chromosome 11q13, encoding the protein bestrophin, was
identified. The function of bestrophin is unknown. To facilitate
studies of bestrophin, we produced both rabbit polyclonal and mouse
monoclonal antibodies that proved useful for Western blotting,
immunoprecipitation, and immunocytochemistry. To characterize
bestrophin, we initially probed the retinal pigment epithelium
(RPE)-derived cell lines ARPE-19, D407, and RPE-J. All of the cell
lines expressed bestrophin mRNA by reverse transcription-PCR, but not
on Western blots. Bestrophin in human RPE partitioned in the detergent
phase during Triton X-114 extraction and could be modified by biotin in
intact cells, indicative of a plasma membrane localization.
Immunocytochemical staining of macaque and porcine eyes indicated that
bestrophin is localized at the basolateral plasma membrane of RPE
cells. When expressed in RPE-J cells by adenovirus-mediated gene
transfer, bestrophin again was determined by confocal microscopy and
cell surface biotinylation to be a basolateral plasma membrane protein.
The basolateral plasma membrane localization of bestrophin suggests the
possibility that bestrophin plays a role in generating the altered
electrooculogram of individuals with Best disease.
Neuroglobin is up-regulated by and protects neurons from hypoxic-ischemic injury
Authors: Sun Yunjuan , Jin Kunlin , Mao Xiao Ou , Zhu Yonghua , Greenberg David A.
Published: July 2002

Globins are oxygen-binding heme proteins present in bacteria,
protists, fungi, plants, and animals. Their functions have diverged
widely in evolution, and include binding, transport, scavenging,
detoxification, and sensing of gases like oxygen, nitric oxide, and
carbon monoxide. Neuroglobin (Ngb) is a recently discovered monomeric
globin with high affinity for oxygen and preferential localization to
vertebrate brain. No function for Ngb is known, but its affinity for
oxygen and its expression in cerebral neurons suggest a role in
neuronal responses to hypoxia or ischemia. Here we report that Ngb
expression is increased by neuronal hypoxia
in vitro
and
focal cerebral ischemia
in vivo
, and that neuronal
survival after hypoxia is reduced by inhibiting Ngb expression with an
antisense oligodeoxynucleotide and enhanced by Ngb overexpression. Both
induction of Ngb and its protective effect show specificity for hypoxia
over other stressors. We conclude that hypoxia-inducible Ngb expression
helps promote neuronal survival from hypoxic-ischemic insults.
Quantal acetylcholine release induced by mediatophore transfection.
Authors: Falk-Vairant J , Corrèges P , Eder-Colli L , Salem N , Roulet E , Bloc A , Meunier F , Lesbats B , Loctin F , Synguelakis M , Israel M , Dunant Y
Published: July 2002

Mediatophore is a protein of approximately 200 kDa able to translocate acetylcholine in response to calcium. It was purified from the presynaptic plasma membranes of the electric organ nerve terminals. Mediatophore is a homooligomer of a 16-kDa subunit, homologous to the proteolipid of V-ATPase. Cells of the N18TG-2 neuronal line are not able to produce quantal acetylcholine release. We show here that transfection of N18TG-2 cells with a plasmid encoding the mediatophore subunit restored calcium-dependent release. The essential feature of such a release was its quantal nature, similar to what is observed in situ in cholinergic synapses from which mediatophore was purified.
The timing of synaptic vesicle endocytosis.
Authors: Ryan T A , Smith S J , Reuter H
Published: July 2002

Alternative models to describe the endocytosis phase of synaptic vesicle recycling are associated with time scales of vesicle recovery ranging from milliseconds to tens of seconds. There have been suggestions that one of the major models, envisioned as a slow process that occurs only after complete fusion of the vesicle membrane with the neurolemma, might be applicable only under conditions of heavy, nonphysiological stimulation. Using FM 1-43 and similar fluorescent probes to label recycling synaptic vesicles in rat hippocampal neurons, we have measured the kinetics of endocytosis with a wide range of action-potential-driven exocytotic loads. Our results indicate that when either 5% or 25% of the vesicle pool is used, vesicles are recovered with a half-time on the order of 20 s (24 degrees C). This endocytosis rate was not influenced by operations designed to alter intracellular Ca2+ during membrane retrieval, suggesting that residual Ca2+ after strong stimuli probably does not greatly retard endocytosis. Finally, we have shown that vesicle-destaining kinetics are not strongly influenced by the substantially differing rates at which two marker dyes tested dissociate from membranes. This observation suggests that vesicles remain open long enough for essentially complete dissociation of even the slower dye (a few seconds) or, alternatively, that both dyes readily escape vesicle membrane by lateral diffusion through any exocytotic opening. These data seem most consistent with applicability of the slow-endocytosis, complete-fusion model at low as well as high levels of exocytosis.
Isolation of an immunodominant viral peptide that is endogenously bound to the stress protein GP96/GRP94.
Authors: Nieland T J , Tan M C , Monne-van Muijen M , Koning F , Kruisbeek A M , van Bleek G M
Published: July 2002

Heat shock protein gp96 primes class I restricted cytotoxic T cells against antigens present in the cells from which it was isolated. Moreover, gp96 derived from certain tumors functions as an effective vaccine, causing complete tumor regressions in in vivo tumor challenge protocols. Because tumor-derived gp96 did not differ from gp96 isolated from normal tissues, a role for gp96 as a peptide carrier has been proposed. To test this hypothesis, we analyzed whether such an association of antigenic peptides with gp96 occurs in a well-defined viral model system. Here we present the full characterization of an antigenic peptide that endogenously associates with the stress protein gp96 in cells infected with vesicular stomatitis virus (VSV). This peptide is identical to the immunodominant peptide of VSV, which is also naturally presented by H-2Kb major histocompatibility complex class I molecules. This peptide associates with gp96 in VSV-infected cells regardless of the major histocompatibility com- plex haplotype of the cell. Our observations provide a biochemical basis for the vaccine function of gp96.
Molecular control of vertebrate iron metabolism: mRNA-based regulatory circuits operated by iron, nitric oxide, and oxidative stress.
Authors: Hentze M W , Kühn L C
Published: July 2002

As an essential nutrient and a potential toxin, iron poses an exquisite regulatory problem in biology and medicine. At the cellular level, the basic molecular framework for the regulation of iron uptake, storage, and utilization has been defined. Two cytoplasmic RNA-binding proteins, iron-regulatory protein-1 (IRP-1) and IRP-2, respond to changes in cellular iron availability and coordinate the expression of mRNAs that harbor IRP-binding sites, iron-responsive elements (IREs). Nitric oxide (NO) and oxidative stress in the form of H2O2 also signal to IRPs and thereby influence cellular iron metabolism. The recent discovery of two IRE-regulated mRNAs encoding enzymes of the mitochondrial citric acid cycle may represent the beginnings of elucidating regulatory coupling between iron and energy metabolism. In addition to providing insights into the regulation of iron metabolism and its connections with other cellular pathways, the IRE/IRP system has emerged as a prime example for the understanding of translational regulation and mRNA stability control. Finally, IRP-1 has highlighted an unexpected role for iron sulfur clusters as post-translational regulatory switches.
Isolated rat hepatocytes can signal to other hepatocytes and bile duct cells by release of nucleotides.
Authors: Schlosser S F , Burgstahler A D , Nathanson M H
Published: July 2002

Intercellular communication among certain cell types can occur via ATP secretion, which leads to stimulation of nucleotide receptors on target cells. In epithelial cells, however, intercellular communication is thought to occur instead via gap junctions. Here we examined whether one epithelial cell type, hepatocytes, can also communicate via nucleotide secretion. The effects on cytosolic Ca2+ ([Ca2+]i) of mechanical stimulation, including microinjection, were examined in isolated rat hepatocytes and in isolated bile duct units using confocal fluorescence video microscopy. Mechanical stimulation of a single hepatocyte evoked an increase in [Ca2+]i in the stimulated cell plus an unexpected [Ca2+]i rise in neighboring noncontacting hepatocytes. Perifusion with ATP before mechanical stimulation suppressed the [Ca2+]i increase, but pretreatment with phenylephrine did not. The P2 receptor antagonist suramin inhibited these intercellular [Ca2+]i signals. The ATP/ADPase apyrase reversibly inhibited the [Ca2+]i rise induced by mechanical stimulation, and did not block vasopressin-induced [Ca2+]i signals. Mechanical stimulation of hepatocytes also induced a [Ca2+]i increase in cocultured isolated bile duct units, and this [Ca2+]i increase was inhibited by apyrase as well. Finally, this form of [Ca2+]i signaling could be elicited in the presence of propidium iodide without nuclear labeling by that dye, indicating that this phenomenon does not depend on disruption of the stimulated cell. Thus, mechanical stimulation of isolated hepatocytes, including by microinjection, can evoke [Ca2+]i signals in the stimulated cell as well as in neighboring noncontacting hepatocytes and bile duct epithelia. This signaling is mediated by release of ATP or other nucleotides into the extracellular space. This is an important technical consideration given the widespread use of microinjection techniques for examining mechanisms of signal transduction. Moreover, the evidence provided suggests a novel paracrine signaling pathway for epithelia, which previously were thought to communicate exclusively via gap junctions.
The dwarf-1 (dt) Mutant of Zea mays blocks three steps in the gibberellin-biosynthetic pathway.
Authors: Spray C R , Kobayashi M , Suzuki Y , Phinney B O , Gaskin P , MacMillan J
Published: July 2002

In plants, gibberellin (GA)-responding mutants have been used as tools to identify the genes that control specific steps in the GA-biosynthetic pathway. They have also been used to determine which native GAs are active per se, i.e., further metabolism is not necessary for bioactivity. We present metabolic evidence that the D1 gene of maize (Zea mays L.) controls the three biosynthetic steps: GA20 to GA1, Ga20 to GA5, and GA5 to GA3. We also present evidence that three gibberellins, GA1, GA5, and GA3, have per se activity in stimulating shoot elongation in maize. The metabolic evidence comes from the injection of [17-13C,3H]GA20 and [17-13C,3H]GA5 into seedlings of d1 and controls (normal and d5), followed by isolation and identification of the 13C-labeled metabolites by full-scan GC-MS and Kovats retention index. For the controls, GA20 was metabolized to GA1,GA3, and GA5; GA5 was metabolized to GA3. For the d1 mutant, GA20 was not metabolized to GA1, GA3, or to GA5, and GA5 was not metabolized to GA3. The bioassay evidence is based on dosage response curves using d1 seedlings for assay. GA1, GA3, and GA5 had similar bioactivities, and they were 10-times more active than GA20.
Immunocytochemical localization of acyl-lipid desaturases in cyanobacterial cells: evidence that both thylakoid membranes and cytoplasmic membranes are sites of lipid desaturation.
Authors: Mustardy L , Los D A , Gombos Z , Murata N
Published: July 2002

There are four acyl-lipid desaturases in the cyanobacterium Synechocystis sp. PCC 6803. Each of these desaturases introduces a double bond at a specific position, such as the Delta6, Delta9, Delta12, or omicron3 position, in C18 fatty acids. The localization of the desaturases in cyanobacterial cells was examined immunocytochemically with antibodies raised against synthetic oligopeptides that corresponded to the carboxyl-terminal regions of the desaturases. All four desaturases appeared to be located in the regions of both the cytoplasmic and the thylakoid membranes. These findings suggest that fatty acid desaturation of membrane lipids takes place in the thylakoid membranes as well as in the cytoplasmic membranes.
Induction of a physiological memory in the cerebral cortex by stimulation of the nucleus basalis.
Authors: Bakin J S , Weinberger N M
Published: July 2002

Auditory cortical receptive field plasticity produced during behavioral learning may be considered to constitute "physiological memory" because it has major characteristics of behavioral memory: associativity, specificity, rapid acquisition, and long-term retention. To investigate basal forebrain mechanisms in receptive field plasticity, we paired a tone with stimulation of the nucleus basalis, the main subcortical source of cortical acetylcholine, in the adult guinea pig. Nucleus basalis stimulation produced electroencephalogram desynchronization that was blocked by systemic and cortical atropine. Paired tone/nucleus basalis stimulation, but not unpaired stimulation, induced receptive field plasticity similar to that produced by behavioral learning. Thus paired activation of the nucleus basalis is sufficient to induce receptive field plasticity, possibly via cholinergic actions in the cortex.
T-cell alpha beta + and gamma delta + deficient mice display abnormal but distinct phenotypes toward a natural, widespread infection of the intestinal epithelium.
Authors: Roberts S J , Smith A L , West A B , Wen L , Findly R C , Owen M J , Hayday A C
Published: July 2002

Vertebrate immune systems contain T cells bearing either alpha beta or gamma delta T-cell antigen receptors (TCRs). alpha beta T cells perform all well-characterized T-cell effector functions, while the biological functions of gamma delta + cells remain unclear. Of particular interest is the role of gamma delta + cells during epithelial infections, since gamma delta + cells are commonly abundant within epithelia. Eimeria spp. are intracellular protozoa that infect epithelia of most vertebrates, causing coccidiosis. This study shows that in response to Eimeria vermiformis, mice lacking alpha beta T cells display defects in protective immunity, while mice lacking gamma delta + cells display exaggerated intestinal damage, apparently due to a failure to regulate the consequences of the alpha beta T cell response. An immuno-downregulatory role during infection, and during autoimmune disease, may be a general one for gamma delta + cells.
Two glucose transporters in Saccharomyces cerevisiae are glucose sensors that generate a signal for induction of gene expression.
Authors: Ozcan S , Dover J , Rosenwald A G , Wölfl S , Johnston M
Published: July 2002

Glucose is the preferred carbon source for most eukaryotic cells and has profound effects on many cellular functions. How cells sense glucose and transduce a signal into the cell is a fundamental, unanswered question. Here we describe evidence that two unusual glucose transporters in the yeast Saccharomyces cerevisiae serve as glucose sensors that generate an intracellular glucose signal. The Snf3p high-affinity glucose transporter appears to function as a low glucose sensor, since it is required for induction of expression of several hexose transporter (HXT) genes, encoding glucose transporters, by low levels of glucose. We have identified another apparent glucose transporter, Rgt2p, that is strikingly similar to Snf3p and is required for maximal induction of gene expression in response to high levels of glucose. This suggests that Rgt2p is a high glucose-sensing counterpart to Snf3p. We identified a dominant mutation in RGT2 that causes constitutive expression of several HXT genes, even in the absence of the inducer glucose. This same mutation introduced into SNF3 also causes glucose-independent expression of HXT genes. Thus, the Rgt2p and Snf3p glucose transporters appear to act as glucose receptors that generate an intracellular glucose signal, suggesting that glucose signaling in yeast is a receptor-mediated process.
Essential role of β-adrenergic receptor kinase 1 in cardiac development and function
Authors: Jaber Mohamed , Koch Walter J. , Rockman Howard , Smith Bradley , Bond Richard A. , Sulik Kathleen K. , Ross John , Lefkowitz Robert J. , Caron Marc G. , Giros Bruno
Published: July 2002

The β-adrenergic receptor kinase 1 (βARK1) is a member of the G
protein-coupled receptor kinase (GRK) family that mediates the
agonist-dependent phosphorylation and desensitization of G
protein-coupled receptors. We have cloned and disrupted the βARK1
gene in mice by homologous recombination. No homozygote
βARK1
−/−
embryos survive beyond gestational day 15.5.
Prior to gestational day 15.5, βARK1
−/−
embryos display
pronounced hypoplasia of the ventricular myocardium essentially
identical to the “thin myocardium syndrome” observed upon gene
inactivation of several transcription factors (RXRα,
N-
myc
, TEF-1, WT-1). Lethality in
βARK1
−/−
embryos is likely due to heart failure as they
exhibit a >70% decrease in cardiac ejection fraction determined by
direct
in utero
intravital microscopy. These results
along with the virtual absence of endogenous GRK activity
in βARK1
−/−
embryos demonstrate that βARK1 appears to
be the predominant GRK in early embryogenesis and that it plays a
fundamental role in cardiac development.
An extracellular signaling component in propagation of astrocytic calcium waves
Authors: Hassinger T. D. , Guthrie P. B. , Atkinson P. B. , Bennett M. V. L. , Kater S. B.
Published: July 2002

Focally evoked calcium waves in astrocyte cultures have been
thought to propagate by gap-junction-mediated intercellular passage of
chemical signal(s). In contrast to this mechanism we observed isolated
astrocytes, which had no physical contact with other astrocytes in the
culture, participating in a calcium wave. This observation requires an
extracellular route of astrocyte signaling. To directly test for
extracellular signaling we made cell-free lanes 10–300 μm wide in
confluent cultures by deleting astrocytes with a glass pipette. After
4–8 hr of recovery, regions of confluent astrocytes separated by lanes
devoid of cells were easily located. Electrical stimulation was used to
initiate calcium waves. Waves crossed narrow (<120 μm) cell-free
lanes in 15 of 36 cases, but failed to cross lanes wider than 120 μm
in eight of eight cases. The probability of crossing narrow lanes was
not correlated with the distance from the stimulation site, suggesting
that cells along the path of the calcium wave release the extracellular
messenger(s). Calculated velocity across the acellular lanes was not
significantly different from velocity through regions of confluent
astrocytes. Focal superfusion altered both the extent and the direction
of calcium waves in confluent regions. These data indicate that
extracellular signals may play a role in astrocyte–astrocyte
communication
in situ
.
E2F1 and E1A(12S) have a homologous activation domain regulated by RB and CBP.
Authors: Trouche D , Kouzarides T
Published: July 2002

The E2F1 transcription factor has a well-characterized activation domain at its C terminus and the E1A protein has a recently defined activation domain at its N terminus. Here we show that these activation domains are highly related in sequence. The sequence homology reflects, at least partly, the conservation of common binding sites for the RB and CBP/p300 proteins, which are preserved in the same relative order along E2F1 and E1A. Furthermore, the interaction of RB and CBP with these two activation domains results in the same functional consequences: RB represses both activation domains, whereas CBP stimulates them. We conclude that the activation domains of E1A(12s) and E2F1 belong to a novel functional class, characterized by specific protein binding sites. The implication of this conservation with respect to E1A-induced stimulation of E2F activity is discussed.