bluuebunny/crossref_metadata_2025 huggingface dataset:
Cloning and characterization of human inducible nitric oxide synthase splice variants: A domain, encoded by exons 8 and 9, is critical for dimerization
Authors: Eissa N. Tony , Yuan Jean W. , Haggerty Cynthia M. , Choo Esther K. , Palmer Celeste D. , Moss Joel
Published: July 2002

The inducible nitric oxide synthase (iNOS) contains an amino-terminal oxygenase domain, a carboxy-terminal reductase domain, and an intervening calmodulin-binding region. For the synthesis of nitric oxide (NO), iNOS is active as a homodimer. The human iNOS mRNA is subject to alternative splicing, including deletion of exons 8 and 9 that encode amino acids 242–335 of the oxygenase domain. In this study, iNOS

8
−

9
−

and full-length iNOS (iNOS
FL
) were cloned from bronchial epithelial cells. Expression of iNOS

8
−

9
−

in 293 cell line resulted in generation of iNOS

8
−

9
−

mRNA and protein but did not lead to NO production. In contrast to iNOS
FL
, iNOS

8
−

9
−

did not form dimers. Similar to iNOS
FL
, iNOS

8
−

9
−

exhibited NADPH-diaphorase activity and contained tightly bound calmodulin, indicating that the reductase and calmodulin-binding domains were functional. To identify sequences in exons 8 and 9 that are critical for dimerization, iNOS
FL
was used to construct 12 mutants, each with deletion of eight residues in the region encoded by exons 8 and 9. In addition, two “control” iNOS deletion mutants were synthesized, lacking either residues 45–52 of the oxygenase domain or residues 1131–1138 of the reductase domain. Whereas both control deletion mutants generated NO and formed dimers, none of the 12 other mutants formed dimers or generated NO. The region encoded by exons 8 and 9 is critical for iNOS dimer formation and NO production but not for reductase activity. This region could be a potential target for therapeutic interventions aimed at inhibiting iNOS dimerization and hence NO synthesis.
The elusive function of metallothioneins
Authors: Palmiter Richard D.
Published: July 2002

Biochemistry and genetics are both required to elucidate the
function of macromolecules. There is no question that
metallothioneins (MTs) have unique biochemical properties, but
genetic experiments have not substantiated the importance of MTs under
physiological conditions. Even after thousands of studies describing
the structure, biochemical characteristics, tissue distribution,
induction, and consequences of genetic disruption and deliberate
overexpression, the evolutionary forces that led to the initial
appearance, gene duplications, and nearly ubiquitous expression of MTs
remain enigmatic.
Evidence that phospholipase A
2
activity is required for Golgi complex and trans Golgi network membrane tubulation
Authors: de Figueiredo Paul , Drecktrah Daniel , Katzenellenbogen John A. , Strang Marian , Brown William J.
Published: July 2002

Membrane tubules of uniform diameter (60–80 nm) and various lengths (up to several micrometers) emanate from elements of the Golgi stack and trans Golgi network (TGN). These organelle membrane tubules are thought to be involved in membrane trafficking and maintenance of Golgi/TGN architecture. The number of these tubules, and their frequency of formation, can be greatly enhanced by the fungal metabolite brefeldin A (BFA), an inhibitor of Golgi/TGN-associated coated vesicle formation. We show here that BFA stimulation of Golgi and TGN membrane tubulation, and the resultant retrograde transport of resident Golgi enzymes to the endoplasmic reticulum, was potently inhibited by a number of membrane-permeant antagonists of phospholipase A
2
(PLA
2
; EC
3.1.1.4
) activity. In addition, PLA
2
inhibitors on their own caused a reversible fragmentation of the Golgi complex into juxtanuclear, stacked cisternal elements. We conclude from these observations that tubulation of Golgi complex and TGN membranes requires a PLA
2
activity, and that this activity may participate not only in Golgi tubule-mediated retrograde trafficking to the endoplasmic reticulum, but also in the maintenance of Golgi complex architecture.
FYB (FYN binding protein) serves as a binding partner for lymphoid protein and FYN kinase substrate SKAP55 and a SKAP55-related protein in T cells
Authors: Liu Junke , Kang Hyun , Raab Monika , da Silva Antonio J. , Kraeft Stine-Kathrein , Rudd Christopher E.
Published: July 2002

TcRζ/CD3 ligation initiates a signaling cascade involving CD4/CD8-p56lck, p59fyn, and ZAP-70, as well as lymphoid downstream proteins VAV, SLP-76, and FYB/SLAP. A current question concerns the nature of the downstream binding partner(s) of FYB in T cells. In this study, using a two-hybrid screen with FYB as bait, we have identified eight clones, four of which correspond to the recently published lymphoid protein SKAP55, and two which correspond to a related protein with some 44% homology to SKAP55 (termed SKAP55-related protein, SKAP55R). The SKAP55 clones showed only minor differences (two substitutions and one residue deletion) from SKAP55. SKAP55R has the same overall structure as SKAP55 except for the presence of a unique N terminus with a well-defined coiled-coil domain. Both SKAP55 and SKAP55R were found to bind FYB through their SH3 domains and to act as substrates for the FYN kinase in T cells. Furthermore, immunofluorescence confocal microscopy showed that FYB and SKAP55 colocalize in the perinuclear region of cells. SKAP55 also colocalizes with another FYB binding protein, SLP-76. Taken together, these observations demonstrate that FYB is part of an interactive matrix with SKAP55 and a SKAP55-related protein.
Flt-1 lacking the tyrosine kinase domain is sufficient for normal development and angiogenesis in mice
Authors: Hiratsuka Sachie , Minowa Osamu , Kuno Junko , Noda Tetsuo , Shibuya Masabumi
Published: July 2002

Receptor tyrosine kinases Flt-1 and Flk-1/KDR, and their ligand, the vascular endothelial growth factor (VEGF), were shown to be essential for angiogenesis in the mouse embryo by gene targeting. Flk-1/KDR null mutant mice exhibited impaired endothelial and hematopoietic cell development. On the other hand, Flt-1 null mutation resulted in early embryonic death at embryonic day 8.5, showing disorganization of blood vessels, such as overgrowth of endothelial cells. Flt-1 differs from Flk-1 in that it displays a higher affinity for VEGF but lower kinase activity, suggesting the importance of its extracellular domain. To examine the biological role of Flt-1 in embryonic development and vascular formation, we deleted the kinase domain without affecting the ligand binding region. Flt-1 tyrosine kinase-deficient homozygous mice (

flt-1
TK−/−

) developed normal vessels and survived. However, VEGF-induced macrophage migration was strongly suppressed in

flt-1
TK−/−

mice. These results indicate that Flt-1 without tyrosine kinase domain is sufficient to allow embryonic development with normal angiogenesis, and that a receptor tyrosine kinase plays a main biological role as a ligand-binding molecule.
Cooperation and self-interest: Pareto-inefficiency of Nash equilibria in finite random games
Authors: Cohen Joel E.
Published: July 2002

The relative merits of cooperation and self-interest in an ensemble of strategic interactions can be investigated by using finite random games. In finite random games, finitely many players have finite numbers of actions and independently and identically distributed (iid) random payoffs with continuous distribution functions. In each realization, players are shown the values of all payoffs and then choose their strategies simultaneously. Noncooperative self-interest is modeled by Nash equilibrium (NE). Cooperation is advantageous when a NE is Pareto-inefficient. In ordinal games, the numerical value of the payoff function gives each player’s ordinal ranking of payoffs. For a fixed number of players, as the number of actions of any player increases, the conditional probability that a pure strategic profile is not pure Pareto-optimal, given that it is a pure NE, apparently increases, but is bounded above strictly below 1. In games with transferable utility, the numerical payoff values may be averaged across actions (so that mixed NEs are meaningful) and added across players. In simulations of two-player games when both players have small, equal numbers of actions, as the number of actions increases, the probability that a NE (pure and mixed) attains the cooperative maximum declines rapidly; the gain from cooperation relative to the Nash high value decreases; and the gain from cooperation relative to the Nash low value rises dramatically. In the cases studied here, with an increasing number of actions, cooperation is increasingly likely to become advantageous compared with pure self-interest, but self-interest can achieve all that cooperation could achieve in a nonnegligible fraction of cases. These results can be interpreted in terms of cooperation in societies and mutualism in biology.
Parathyroid hormone-related protein is required for tooth eruption
Authors: Philbrick William M. , Dreyer Barbara E. , Nakchbandi Inaam A. , Karaplis Andrew C.
Published: July 2002

Parathyroid hormone (PTH)-related protein (PTHrP)-knockout mice die at birth with a chondrodystrophic phenotype characterized by premature chondrocyte differentiation and accelerated bone formation, whereas overexpression of PTHrP in the chondrocytes of transgenic mice produces a delay in chondrocyte maturation and endochondral ossification. Replacement of PTHrP expression in the chondrocytes of PTHrP-knockout mice using a procollagen II-driven transgene results in the correction of the lethal skeletal abnormalities and generates animals that are effectively PTHrP-null in all sites other than cartilage. These rescued PTHrP-knockout mice survive to at least 6 months of age but are small in stature and display a number of developmental defects, including cranial chondrodystrophy and a failure of tooth eruption. Teeth appear to develop normally but become trapped by the surrounding bone and undergo progressive impaction. Localization of PTHrP mRNA during normal tooth development by
in situ
hybridization reveals increasing levels of expression in the enamel epithelium before the formation of the eruption pathway. The type I PTH/PTHrP receptor is expressed in both the adjacent dental mesenchyme and in the alveolar bone. The replacement of PTHrP expression in the enamel epithelium with a keratin 14-driven transgene corrects the defect in bone resorption and restores the normal program of tooth eruption. PTHrP therefore represents an essential signal in the formation of the eruption pathway.
Altered surfactant homeostasis and alveolar type II cell morphology in mice lacking surfactant protein D
Authors: Botas Carlos , Poulain Francis , Akiyama Jennifer , Brown Cindy , Allen Lennell , Goerke Jon , Clements John , Carlson Elaine , Gillespie Anne Marie , Epstein Charles , Hawgood Samuel
Published: July 2002

Surfactant protein D (SP-D) is one of two collectins found in the pulmonary alveolus. On the basis of homology with other collectins, potential functions for SP-D include roles in innate immunity and surfactant metabolism. The SP-D gene was disrupted in embryonic stem cells by homologous recombination to generate mice deficient in SP-D. Mice heterozygous for the mutant SP-D allele had SP-D concentrations that were approximately 50% wild type but no other obvious phenotypic abnormality. Mice totally deficient in SP-D were healthy to 7 months but had a progressive accumulation of surfactant lipids, SP-A, and SP-B in the alveolar space. By 8 weeks the alveolar phospholipid pool was 8-fold higher than wild-type littermates. There was also a 10-fold accumulation of alveolar macrophages in the null mice, and many macrophages were both multinucleated and foamy in appearance. Type II cells in the null mice were hyperplastic and contained giant lamellar bodies. These alterations in surfactant homeostasis were not associated with detectable changes in surfactant surface activity, postnatal respiratory function, or survival. The findings in the SP-D-deficient mice suggest a role for SP-D in surfactant homeostasis.
The neuronal RNA-binding protein Nova-2 is implicated as the autoantigen targeted in POMA patients with dementia
Authors: Yang Yolanda Y. L. , Yin Guang Lin , Darnell Robert B.
Published: July 2002

Paraneoplastic opsoclonus myoclonus ataxia (POMA) is a neurologic disorder thought to be mediated by an autoimmune attack against onconeural disease antigens that are expressed by gynecologic or lung tumors and by neurons. One POMA disease antigen, termed Nova-1, has been identified as a neuron-specific KH-type RNA-binding protein. Nova-1 expression is restricted to specific regions of the central nervous system, primarily the hindbrain and ventral spinal cord, which correlate with the predominantly motor symptoms in POMA. However, POMA antisera recognize antigens that are widely expressed in both caudal and rostral regions of the central nervous system, and some patients develop cognitive symptoms. We have used POMA antisera to clone a cDNA encoding a second POMA disease antigen termed Nova-2. Nova-2 is closely related to Nova-1, and is expressed at high levels in neurons during development and in adulthood, and at lower levels in the adult lung. In the postnatal mouse brain, Nova-2 is expressed in a pattern that is largely reciprocal with Nova-1, including high levels of Nova-2 expression in the neocortex and hippocampus. Functional characterization of Nova-2 in RNA selection and nitrocellulose filter-binding assays reveals that Nova-2 binds RNA with high affinity and with sequence specificity that differs from Nova-1. Our results demonstrate that the immune response in POMA targets a family of highly related sequence-specific neuronal RNA-binding proteins. The expression pattern of the Nova-2 protein is likely to underlie the development of cognitive deficits in some POMA patients.
An empirical explanation of brightness
Authors: Williams S. Mark , McCoy Allison N. , Purves Dale
Published: July 2002

In this second part of our study on the mechanism of perceived brightness, we explore the effects of manipulating three-dimensional geometry. The additional scenes portrayed here demonstrate that the same luminance profile can elicit different sensations of brightness as a function of how the objects in the scene are arranged in space. This further evidence confirms the implication of the scenes presented in the accompanying paper, namely that sensations of relative brightness—including standard demonstrations of simultaneous brightness contrast—cannot arise by computations of local contrast. The most plausible explanation of the full range of perceptual phenomena we have described is an empirical strategy that links the luminance profile in a visual stimulus with an association (the percept) that represents the profile’s most probable real-world source.
Phosphorylation of the rRNA transcription factor upstream binding factor promotes its association with TATA binding protein
Authors: Kihm Anthony J. , Hershey James C. , Haystead Timothy A. J. , Madsen Cort S. , Owens Gary K.
Published: July 2002

rRNA synthesis by RNA polymerase I requires both the promoter selectivity factor 1, which is composed of TATA binding protein (TBP) and three TBP-associated factors, and the activator upstream binding factor (UBF). Whereas there is strong evidence implicating a role for phosphorylation of UBF in the control of growth-induced increases in rRNA transcription, the mechanism of this effect is not known. Results of immunoprecipitation studies with TBP antibodies showed increased recovery of phosphorylated UBF from growth-stimulated smooth muscle cells. Moreover, using an immobilized protein-binding assay, we found that phosphorylation of UBF
in vivo
in response to stimulation with different growth factors or
in vitro
with smooth muscle cell nuclear extract increased its binding to TBP. Finally, we demonstrated that UBF–TBP binding depended on the C-terminal ‘acidic tail’ of UBF that was hyperphosphorylated at multiple serine sites after growth factor stimulation. Results of these studies suggest that phosphorylation of UBF and subsequent binding to TBP represent a key regulatory step in control of growth-induced increases in rRNA synthesis.
Activation of human primary motor cortex during action observation: A neuromagnetic study
Authors: Hari R. , Forss N. , Avikainen S. , Kirveskari E. , Salenius S. , Rizzolatti G.
Published: July 2002

The monkey premotor cortex contains neurons that discharge during action execution and during observation of actions made by others. Transcranial magnetic stimulation experiments suggest that a similar observation/execution matching system also is present in humans. We recorded neuromagnetic oscillatory activity of the human precentral cortex from 10 healthy volunteers while (
i
) they had no task to perform, (
ii
) they were manipulating a small object, and (
iii
) they were observing another individual performing the same task. The left and right median nerves were stimulated alternately (interstimulus interval, 1.5 s) at intensities exceeding motor threshold, and the poststimulus rebound of the rolandic 15- to 25-Hz activity was quantified. In agreement with previous studies, the rebound was strongly suppressed bilaterally during object manipulation. Most interestingly, the rebound also was significantly diminished during action observation (31–46% of the suppression during object manipulation). Control experiments, in which subjects were instructed to observe stationary or moving stimuli, confirmed the specificity of the suppression effect. Because the recorded 15- to 25-Hz activity is known to originate mainly in the precentral motor cortex, we concluded that the human primary motor cortex is activated during observation as well as execution of motor tasks. These findings have implications for a better understanding of the machinery underlying action recognition in humans.
Inhibition of β-catenin-mediated transactivation by cadherin derivatives
Authors: Sadot Einat , Simcha Inbal , Shtutman Michael , Ben-Ze’ev Avri , Geiger Benjamin
Published: July 2002

We studied the effect of N-cadherin, and its free or membrane-anchored cytoplasmic domain, on the level and localization of β-catenin and on its ability to induce lymphocyte enhancer-binding factor 1 (LEF-1)-responsive transactivation. These cadherin derivatives formed complexes with β-catenin and protected it from degradation. N-cadherin directed β-catenin into adherens junctions, and the chimeric protein induced diffuse distribution of β-catenin along the membrane whereas the cytoplasmic domain of N-cadherin colocalized with β-catenin in the nucleus. Cotransfection of β-catenin and LEF-1 into Chinese hamster ovary cells induced transactivation of a LEF-1 reporter, which was blocked by the N-cadherin-derived molecules. Expression of N-cadherin and an interleukin 2 receptor/cadherin chimera in SW480 cells relocated β-catenin from the nucleus to the plasma membrane and reduced transactivation. The cytoplasmic tails of N- or E-cadherin colocalized with β-catenin in the nucleus, and suppressed the constitutive LEF-1-mediated transactivation, by blocking β-catenin–LEF-1 interaction. Moreover, the 72 C-terminal amino acids of N-cadherin stabilized β-catenin and reduced its transactivation potential. These results indicate that β-catenin binding to the cadherin cytoplasmic tail either in the membrane, or in the nucleus, can inhibit β-catenin degradation and efficiently block its transactivation capacity.
A potential role for the nuclear factor of activated T cells family of transcriptional regulatory proteins in adipogenesis
Authors: Ho I-Cheng , Kim John H.-J. , Rooney John W. , Spiegelman Bruce M. , Glimcher Laurie H.
Published: July 2002

NFAT (nuclear factor of activated T cells) is a family of transcription factors implicated in the control of cytokine and early immune response gene expression. Recent studies have pointed to a role for NFAT proteins in gene regulation outside of the immune system. Herein we demonstrate that NFAT proteins are present in 3T3-L1 adipocytes and, upon fat cell differentiation, bind to and transactivate the promoter of the adipocyte-specific gene aP2. Further, fat cell differentiation is inhibited by cyclosporin A, a drug shown to prevent NFAT nuclear localization and hence function. Thus, these data suggest a role for NFAT transcription factors in the regulation of the aP2 gene and in the process of adipocyte differentiation.
Repetitive transcranial magnetic stimulation activates specific regions in rat brain
Authors: Ji Ru-Rong , Schlaepfer Thomas E. , Aizenman Carlos D. , Epstein Charles M. , Qiu Dike , Huang Justin C. , Rupp Fabio
Published: July 2002

Repetitive transcranial magnetic stimulation (rTMS) is a noninvasive technique to induce electric currents in the brain. Although rTMS is being evaluated as a possible alternative to electroconvulsive therapy for the treatment of refractory depression, little is known about the pattern of activation induced in the brain by rTMS. We have compared immediate early gene expression in rat brain after rTMS and electroconvulsive stimulation, a well-established animal model for electroconvulsive therapy. Our result shows that rTMS applied in conditions effective in animal models of depression induces different patterns of immediate-early gene expression than does electroconvulsive stimulation. In particular, rTMS evokes strong neural responses in the paraventricular nucleus of the thalamus (PVT) and in other regions involved in the regulation of circadian rhythms. The response in PVT is independent of the orientation of the stimulation probe relative to the head. Part of this response is likely because of direct activation, as repetitive magnetic stimulation also activates PVT neurons in brain slices.
A multidrug resistance transporter from human MCF-7 breast cancer cells
Authors: Doyle L. Austin , Yang Weidong , Abruzzo Lynne V. , Krogmann Tammy , Gao Yongming , Rishi Arun K. , Ross Douglas D.
Published: July 2002

MCF-7/AdrVp is a multidrug-resistant human breast cancer subline that displays an ATP-dependent reduction in the intracellular accumulation of anthracycline anticancer drugs in the absence of overexpression of known multidrug resistance transporters such as P glycoprotein or the multidrug resistance protein. RNA fingerprinting led to the identification of a 2.4-kb mRNA that is overexpressed in MCF-7/AdrVp cells relative to parental MCF-7 cells. The mRNA encodes a 663-aa member of the ATP-binding cassette superfamily of transporters that we term breast cancer resistance protein (BCRP). Enforced expression of the full-length BCRP cDNA in MCF-7 breast cancer cells confers resistance to mitoxantrone, doxorubicin, and daunorubicin, reduces daunorubicin accumulation and retention, and causes an ATP-dependent enhancement of the efflux of rhodamine 123 in the cloned transfected cells. BCRP is a xenobiotic transporter that appears to play a major role in the multidrug resistance phenotype of MCF-7/AdrVp human breast cancer cells.
Individual variation in neuron number predicts differences in the propensity for avian vocal imitation
Authors: Ward B. C. , Nordeen E. J. , Nordeen K. W.
Published: July 2002

Avian song learning involves memorizing and reproducing song material produced by conspecifics. In several species song repertoire size correlates with the overall volume of two song-related brain regions, the HVc (acronym used as the proper name) and the robust nucleus of the archistriatum (RA). We raised male zebra finches with two adult tutors and found that individual differences in HVc volume and neuron number correlated positively with differences in the number of tutor syllables accurately copied. These results were replicated in a second study. The relationship between RA volume and song learning was similar, but less robust. Importantly, total repertoire size (number of song syllables) did not correlate significantly with anatomical measures of either the HVc or RA. Because previous work suggests that the volume and neuron number of these regions are not regulated by song learning, it is possible that naturally occurring variation in neuron number constrains how much song material can be copied or reproduced.
Frontoparietal cortical networks for directing attention and the eye to visual locations: Identical, independent, or overlapping neural systems?
Authors: Corbetta Maurizio
Published: July 2002

Functional anatomical and single-unit recording studies indicate that a set of neural signals in parietal and frontal cortex mediates the covert allocation of attention to visual locations, as originally proposed by psychological studies. This frontoparietal network is the source of a location bias that interacts with extrastriate regions of the ventral visual system during object analysis to enhance visual processing. The frontoparietal network is not exclusively related to visual attention, but may coincide or overlap with regions involved in oculomotor processing. The relationship between attention and eye movement processes is discussed at the psychological, functional anatomical, and cellular level of analysis.
Parathyroid hormone activation of the 25-hydroxyvitamin D
3
-1α-hydroxylase gene promoter
Authors: Brenza Holly L. , Kimmel-Jehan Christine , Jehan Frederic , Shinki Toshimasa , Wakino Shu , Anazawa Hideharu , Suda Tatsuo , DeLuca Hector F.
Published: July 2002

The DNA flanking the 5′ sequence of the mouse 1α-hydroxylase gene has been cloned and sequenced. A TATA box has been located at −30 bp and aCCAAT box has been located at −79 bp. The gene’s promoter activity has been demonstrated by using a luciferase reporter gene construct transfected into a modified pig kidney cell line, AOK-B50. Parathyroid hormone stimulates this promoter-directed synthesis of luciferase by 17-fold, whereas forskolin stimulates it by 3-fold. The action of parathyroid hormone is concentration-dependent. 1,25-Dihydroxyvitamin D
3
does not suppress basal promoter activity and marginally suppresses parathyroid hormone-driven luciferase reporter activity. The promoter has three potential cAMP-responsive element sites, and two perfect and one imperfect AP-1 sites, while no DR-3 was detected. These results indicate that parathyroid hormone stimulates 25-hydroxyvitamin D
3
-1α-hydroxylase by acting on the promoter of the 1α-hydroxylase gene.
Modification of expression and
cis
-regulation of
Hoxc8
in the evolution of diverged axial morphology
Authors: Belting Heinz-Georg , Shashikant Cooduvalli S. , Ruddle Frank H.
Published: July 2002

Differential
Hox
gene expression between vertebrate species has been implicated in the divergence of axial morphology. To examine this relationship, we have compared expression and transcriptional regulation of
Hoxc8
in chicken and mouse. In both species, expression of
Hoxc8
in the paraxial mesoderm and neural tube is associated with midthoracic and brachial identities, respectively. During embryogenesis, there is a temporal delay in the activation of
Hoxc8
in chicken compared with mouse. As a result, chicken
Hoxc8
expression in the paraxial mesoderm is at a posterior axial level, extending over a smaller domain compared with mouse
Hoxc8
expression. This finding is consistent with a shorter thoracic region in chicken compared with mouse. In addition, the chicken
Hoxc8
early enhancer, differing from its mouse counterpart in only a few specific nucleotides, directs a reporter gene expression to a more posterior domain in transgenic mouse embryos. These findings are consistent with the concept that the diversification of axial morphology has been achieved through changes in
cis
-regulation of developmental control genes.
Synchronicity of frequently sampled, 24-h concentrations of circulating leptin, luteinizing hormone, and estradiol in healthy women
Authors: Licinio Julio , Negrão André B. , Mantzoros Christos , Kaklamani Virginia , Wong Ma-Li , Bongiorno Peter B. , Mulla Abeda , Cearnal Laura , Veldhuis Johannes D. , Flier Jeffrey S. , McCann Samuel M. , Gold Philip W.
Published: July 2002

Leptin, an adipocyte hormone, is a trophic factor for the reproductive system; however, it is still unknown whether there is a dynamic relation between fluctuations in circulating leptin and hypothalamic—pituitary–ovarian (HPO) axis hormones. To test the hypothesis that fluctuations in plasma leptin concentrations are related to the levels of luteinizing hormone (LH) and estradiol, we sampled plasma from six healthy women every 7 min for 24 h during days 8–11 of the menstrual cycle. Cross-correlation analysis throughout the 24-h cycle revealed a relation between release patterns of leptin and LH, with a lag of 42–84 min but no significant cross-correlation between LH and estradiol. The ultradian fluctuations in leptin levels showed pattern synchrony with those of both LH and estradiol as determined by cross-approximate entropy (cross-ApEn). At night, as leptin levels rose to their peak, the pulsatility profiles of LH changed significantly and became synchronous with those of leptin. LH pulses were fewer, of longer duration, higher amplitude, and larger area than during the day. Moreover, the synchronicity of LH and leptin occurred late at night, at which time estradiol and leptin also exhibited significantly stronger pattern coupling than during the day. We propose that leptin may regulate the minute-to-minute oscillations in the levels of LH and estradiol, and that the nocturnal rise in leptin may determine the change in nocturnal LH profile in the mid-to-late follicular phase that precedes ovulation. This may explain the disruption of hypothalamic—pituitary–ovarian function that is characteristic of states of low leptin release, such as anorexia nervosa and cachexia.
Membrane physical state controls the signaling mechanism of the heat shock response in
Synechocystis
PCC 6803: Identification of
hsp17
as a “fluidity gene”
Authors: Horváth Ibolya , Glatz Attila , Varvasovszki Viktória , Török Zsolt , Páli Tibor , Balogh Gábor , Kovács Eszter , Nádasdi Levente , Benkö Sándor , Joó Ferenc , Vígh László
Published: July 2002

The fluidity of
Synechocystis
membranes was adjusted
in vivo
by temperature acclimation, addition of fluidizer agent benzyl alcohol, or catalytic lipid hydrogenation specific to plasma membranes. The reduced membrane physical order in thylakoids obtained by either downshifting growth temperature or administration of benzyl alcohol was paralleled with enhanced thermosensitivity of the photosynthetic membrane. Simultaneously, the stress-sensing system leading to the cellular heat shock (HS) response also has been altered. There was a close correlation between thylakoid fluidity levels, monitored by steady-state 1,6-diphenyl-1,3,5-hexatriene anisotropy, and threshold temperatures required for maximal activation of all of the HS-inducible genes investigated, including
dnaK
,
groESL
,
cpn60
, and
hsp17
. The causal relationship between the pre-existing thylakoid physical order and temperature set point of both the transcriptional activation and the
de novo
protein synthesis was the most striking for the 17-kDa HS protein (HSP17) associated mostly with the thylakoid membranes. These findings together with the fact that the
in vivo
modulation of lipid saturation within cytoplasmic membrane had no effect on HS response suggest that thylakoid acts as a cellular thermometer where thermal stress is sensed and transduced into a cellular signal leading to the activation of HS genes.
Prolonged delivery of brain-derived neurotrophic factor by adenovirus-infected Müller cells temporarily rescues injured retinal ganglion cells
Authors: Di Polo Adriana , Aigner Ludwig J. , Dunn Robert J. , Bray Garth M. , Aguayo Albert J.
Published: July 2002

In this study, we demonstrate that: (
i
) injection of an adenovirus (Ad) vector containing the brain-derived neurotrophic factor (BDNF) gene (Ad.BDNF) into the vitreous chamber of adult rats results in selective transgene expression by Müller cells; (
ii
)
in vitro
, Müller cells infected with Ad.BDNF secrete BDNF that enhances neuronal survival; (
iii
)
in vivo
, Ad-mediated expression of functional BDNF by Müller cells, temporarily extends the survival of axotomized retinal ganglion cells (RGCs); 16 days after axotomy, injured retinas treated with Ad.BDNF showed a 4.5-fold increase in surviving RGCs compared with control retinas; (
iv
) the transient expression of the BDNF transgene, which lasted ≈10 days, can be prolonged with immunosuppression for at least 30 days, and such Ad-mediated BDNF remains biologically active, (
v
) persistent expression of BDNF by infected Müller cells does not further enhance the survival of injured RGCs, indicating that the effect of this neurotrophin on RGC survival is limited by changes induced by the lesion within 10–16 days after optic nerve transection rather than the availability of BDNF. Thus, Ad-transduced Müller cells are a novel pathway for sustained delivery of BDNF to acutely-injured RGCs. Because these cells span the entire thickness of the retina, Ad-mediated gene delivery to Müller cells may also be useful to influence photoreceptors and other retinal neurons.
Stress facilitates classical conditioning in males, but impairs classical conditioning in females through activational effects of ovarian hormones
Authors: Wood Gwendolyn E. , Shors Tracey J.
Published: July 2002

Exposure to restraint and brief intermittent tailshocks facilitates associative learning of the classical conditioned eyeblink response in male rats. Based on evidence of sex differences in learning and responses to stressful events, we investigated sexually dimorphic effects of a stressor of restraint and intermittent tailshock on classical eyeblink conditioning 24 h after stressor cessation. Our results indicate that exposure to the acute stressor had diametrically opposed effects on the rate of acquisition of the conditioned response in male vs. female rats. Exposure to the stressor facilitated acquisition of the conditioned response in males, whereas exposure to the same stressful event dramatically impaired acquisition in females. We further demonstrate that the stress-induced impairment in female conditioning is dependent on the presence of ovarian hormones. Conditioning of stressed sham-ovariectomized females was significantly impaired relative to the unstressed controls, whereas conditioning in stressed ovariectomized females was not impaired. We present additional evidence that estrogen mediates the stress-induced impairment in female acquisition. Females administered sesame oil vehicle and then stressed were significantly impaired relative to their unstressed controls, whereas females administered the estrogen antagonist tamoxifen prior to stress were not impaired. In summary, these results indicate that exposure to the same aversive event can induce opposite behavioral responses in males vs. females. These effects underscore sex differences in associative learning and emotional responding, and implicate estrogen in the underlying neuronal mechanism.
Breakers of advanced glycation end products restore large artery properties in experimental diabetes
Authors: Wolffenbuttel Bruce H. R. , Boulanger Chantal M. , Crijns Francy R. L. , Huijberts Maya S. P. , Poitevin Pierre , Swennen Geertje N. M. , Vasan Sara , Egan John J. , Ulrich Peter , Cerami Anthony , Lévy Bernard I.
Published: July 2002

Glucose and other reducing sugars react with proteins by a nonenzymatic, posttranslational modification process called nonenzymatic glycation. The formation of advanced glycation end products (AGEs) on connective tissue and matrix components accounts largely for the increase in collagen crosslinking that accompanies normal aging and which occurs at an accelerated rate in diabetes, leading to an increase in arterial stiffness. A new class of AGE crosslink “breakers” reacts with and cleaves these covalent, AGE-derived protein crosslinks. Treatment of rats with streptozotocin-induced diabetes with the AGE-breaker ALT-711 for 1–3 weeks reversed the diabetes-induced increase of large artery stiffness as measured by systemic arterial compliance, aortic impedance, and carotid artery compliance and distensibility. These findings will have considerable implications for the treatment of patients with diabetes-related complications and aging.
Bax directly induces release of cytochrome
c
from isolated mitochondria
Authors: Jürgensmeier Juliane M. , Xie Zhihua , Deveraux Quinn , Ellerby Lisa , Bredesen Dale , Reed John C.
Published: July 2002

Bax is a pro-apoptotic member of the Bcl-2 protein family that resides in the outer mitochondrial membrane. It is controversial whether Bax promotes cell death directly through its putative function as a channel protein versus indirectly by inhibiting cellular regulators of the cell death proteases (caspases). We show here that addition of submicromolar amounts of recombinant Bax protein to isolated mitochondria can induce cytochrome
c
(Cyt
c
) release, whereas a peptide representing the Bax BH3 domain was inactive. When placed into purified cytosol, neither mitochondria nor Bax individually induced proteolytic processing and activation of caspases. In contrast, the combination of Bax and mitochondria triggered release of Cyt
c
from mitochondria and induced caspase activation in cytosols. Supernatants from Bax-treated mitochondria also induced caspase processing and activation. Recombinant Bcl-X
L
protein abrogated Bax-induced release of Cyt
c
from isolated mitochondria and prevented caspase activation. In contrast, the broad-specificity caspase inhibitor benzyloxycarbonyl-valinyl-alaninyl-aspartyl-(
0
-methyl)-fluoromethylketone (zVAD-fmk) and the caspase-inhibiting protein X-IAP had no effect on Bax-induced release of Cyt
c
from mitochondria
in vitro
but prevented the subsequent activation of caspases in cytosolic extracts. Unlike Ca
2+
, a classical inducer of mitochondrial permeability transition, Bax did not induce swelling of mitochondria
in vitro
. Because the organellar swelling caused by permeability transition causes outer membrane rupture, the findings, therefore, dissociate these two events, implying that Bax uses an alternative mechanism for triggering release of Cyt
c
from mitochondria.
Maturation of the tyrosine kinase c-src as a kinase and as a substrate depends on the molecular chaperone Hsp90
Authors: Xu Yang , Singer Mike A. , Lindquist Susan
Published: July 2002

Although Hsp90 displays general chaperone activity
in vitro
, few substrates of the chaperone have been identified
in vivo
, and the characteristics that render these substrates dependent on Hsp90 remain elusive. To investigate this issue, we exploited a paradoxical observation: several unrelated oncogenic viral tyrosine kinases, including v-src, attain their native conformation after association with Hsp90, yet their nearly identical cellular homologs interact only weakly with the chaperone. It has been controversial whether Hsp90 is vital for normal maturation of the cellular kinases or is simply binding a misfolded subfraction of the proteins. By modulating Hsp90 levels in
Saccharomyces cerevisiae
, we determined that Hsp90 is indeed necessary for the maturation of c-src (the normal homolog of v-src). c-src maturation is, however, less sensitive to Hsp90 perturbations than is v-src maturation. Dependence of the two proteins on Hsp90 does not correspond to their relative efficiency in reaching their final destination (the plasma membrane); we observed that in yeast, unlike in vertebrate cells, neither c-src nor v-src concentrate in the membrane. Expression of different v/c-src chimeras in cells carrying wild-type or temperature-sensitive Hsp90 alleles revealed that the difference between the proteins instead arises from multiple, naturally occurring mutations in the C-terminal region of v-src.
Transient and stable gene expression in mammalian cells transduced with a recombinant baculovirus vector
Authors: Condreay J. Patrick , Witherspoon Sam M. , Clay William C. , Kost Thomas A.
Published: July 2002

Recombinant baculoviruses can serve as gene-transfer vehicles for transient expression of recombinant proteins in a wide range of mammalian cell types. Furthermore, by inclusion of a dominant selectable marker in the viral vector, cell lines can be derived that stably express recombinant genes. A virus was constructed containing two expression cassettes controlled by constitutive mammalian promoters: the cytomegalovirus immediate early promoter/enhancer directing expression of green fluorescent protein and the simian virus 40 (SV40) early promoter controlling neomycin phosphotransferase II. Using this virus, efficient gene delivery and expression was observed and measured in numerous cell types of human, primate, and rodent origin. In addition to commonly used transformed cell lines such as HeLa, CHO, Cos-7, and 293, this list includes primary human keratinocytes and bone marrow fibroblasts. In all cases, addition of butyrate or trichostatin A (a selective histone deacetylase inhibitor) to transduced cells markedly enhanced the levels of reporter protein expression observed. When transduced cells are put under selection with the antibiotic G418, cell lines can be obtained at high frequency that stably maintain the expression cassettes of the vector DNA and exhibit stable, high-level expression of the reporter gene. Stably transduced derivatives have been selected from a substantial number of different cell types, suggesting that stable lines can be derived from any cell type that exhibits transient expression.
Interaction of rat hormone-sensitive lipase with adipocyte lipid-binding protein
Authors: Shen Wen-Jun , Sridhar Kunju , Bernlohr David A. , Kraemer Fredric B.
Published: July 2002

Hormone-sensitive lipase (HSL) is a cytosolic neutral lipase that functions as the rate-limiting enzyme for the mobilization of free fatty acids in adipose tissue. By using the yeast two-hybrid system to examine the potential interaction of HSL with other cellular proteins, evidence is provided to demonstrate a direct interaction of HSL with adipocyte lipid-binding protein (ALBP), a member of the family of intracellular lipid-binding proteins that binds fatty acids, retinoids, and other hydrophobic ligands. The interaction was demonstrated
in vitro
by the binding of ALBP to HSL translated
in vitro
, to HSL in extracts of HSL overexpressing Chinese hamster ovary (CHO) cells, and to HSL in extracts of rat adipose tissue. Finally, the presence of ALBP was documented in immune complexes from rat adipose tissue immunoprecipitated with anti-HSL antibodies. The HSL–ALBP interaction was mapped to an N-terminal 300-aa region of HSL that is distinct from the C-terminal catalytic domain. These results suggest that HSL-derived fatty acids are bound by ALBP to facilitate intracellular trafficking of hydrophobic lipids.
Structural determinants for recognition and translocation by the anandamide transporter
Authors: Piomelli D. , Beltramo M. , Glasnapp S. , Lin S. Y. , Goutopoulos A. , Xie Xiang-Qun , Makriyannis A.
Published: July 2002

The biological actions of anandamide (arachidonylethanolamide), an endogenous cannabinoid lipid, are terminated by a two-step inactivation process consisting of carrier-mediated uptake and intracellular hydrolysis. Anandamide uptake in neurons and astrocytes is mediated by a high-affinity, Na
+
-independent transporter that is selectively inhibited by
N
-(4-hydroxyphenyl)-arachidonamide (AM404). In the present study, we examined the structural determinants governing recognition and translocation of substrates by the anandamide transporter constitutively expressed in a human astrocytoma cell line. Competition experiments with a select group of analogs suggest that substrate recognition by the transporter is favored by a polar nonionizable head group of defined stereochemical configuration containing a hydroxyl moiety at its distal end. The secondary carboxamide group interacts favorably with the transporter, but may be replaced with either a tertiary amide or an ester, suggesting that it may serve as hydrogen acceptor. Thus, 2-arachidonylglycerol, a putative endogenous cannabinoid ester, also may serve as a substrate for the transporter. Substrate recognition requires the presence of at least one cis double bond situated at the middle of the fatty acid carbon chain, indicating a preference for ligands whose hydrophobic tail can adopt a bent U-shaped conformation. On the other hand, uptake experiments with radioactively labeled substrates show that no fewer than four cis nonconjugated double bonds are required for optimal translocation across the cell membrane, suggesting that substrates are transported in a folded hairpin conformation. These results outline the general structural requisites for anandamide transport and may assist in the development of selective inhibitors with potential clinical applications.
X inactivation and somatic cell selection rescue female mice carrying a
Piga
-null mutation
Authors: Keller Peter , Tremml Gabi , Rosti Vittorio , Bessler Monica
Published: July 2002

A somatic mutation in the X linked
PIGA
gene is responsible for the deficiency of glycosyl phosphatidylinositol (GPI)-anchored proteins on blood cells from patients with paroxysmal nocturnal hemoglobinuria. No inherited form of GPI-anchor deficiency has been described. Because conventional
Piga
gene knockout is associated with high embryonic lethality in chimeric mice, we used the Cre/loxP system. We generated mice in which two loxP sites flank part of
Piga
exon 2. After crossbreeding with female mice of the
EIIa-cre
strain, the floxed allele undergoes Cre-mediated recombination with high efficiency during early embryonic development. Because of X chromosome inactivation, female offspring are mosaic for cells that express or lack GPI-linked proteins. Analysis of mosaic mice showed that in heart, lung, kidney, brain, and liver, mainly wild-type
Piga
is active, suggesting that these tissues require GPI-linked proteins. The salient exceptions were spleen, thymus, and red blood cells, which had almost equal numbers of cells expressing the wild-type or the recombined allele, implying that GPI-linked proteins are not essential for the derivation of these tissues. PIGA(−) cells had no growth advantage, suggesting that other factors are needed for their clonal dominance in patients with paroxysmal nocturnal hemoglobinuria.
The genetic mediation of individual differences in sensitivity to pain and its inhibition
Authors: Mogil Jeffrey S.
Published: July 2002

The underlying bases of the considerable interindividual variability in pain-related traits are starting to be revealed. Although the relative importance of genes versus experience in human pain perception remains unclear, rodent populations display large and heritable differences in both nociceptive and analgesic sensitivity. The identification and characterization of particularly divergent populations provides a powerful initial step in the genetic analysis of pain, because these models can be exploited to identify genes contributing to the behavior-level variability. Ultimately, DNA sequence differences representing the differential alleles at pain-relevant genes can be identified. Thus, by using a combination of “top-down” and “bottom-up” strategies, we are now able to genetically dissect even complex biological traits like pain. The present review summarizes the current progress toward these ends in both humans and rodents.