bluuebunny/crossref_metadata_2025 huggingface dataset:
Protein-bound acrolein: Potential markers for oxidative stress
Authors: Uchida Koji , Kanematsu Masamichi , Sakai Kensuke , Matsuda Tsukasa , Hattori Nobutaka , Mizuno Yoshikuni , Suzuki Daisuke , Miyata Toshio , Noguchi Noriko , Niki Etsuo , Osawa Toshihiko
Published: July 2002

Acrolein (CH
2
=CH—CHO) is known as a ubiquitous pollutant in the environment. Here we show that this notorious aldehyde is not just a pollutant, but also a lipid peroxidation product that could be ubiquitously generated in biological systems. Upon incubation with BSA, acrolein was rapidly incorporated into the protein and generated the protein-linked carbonyl derivative, a putative marker of oxidatively modified proteins under oxidative stress. To verify the presence of protein-bound acrolein
in vivo
, the mAb (mAb5F6) against the acrolein-modified keyhole limpet hemocyanin was raised. It was found that the acrolein-lysine adduct,
N
ɛ
-(3-formyl-3,4-dehydropiperidino)lysine, constitutes an epitope of the antibody. Immunohistochemical analysis of atherosclerotic lesions from a human aorta demonstrated that antigenic materials recognized by mAb5F6 indeed constituted the lesions, in which intense positivity was associated primarily with macrophage-derived foam cells and the thickening neointima of arterial walls. The observations that (
i
) oxidative modification of low-density lipoprotein with Cu
2+
generated the acrolein-low-density lipoprotein adducts and (
ii
) the iron-catalyzed oxidation of arachidonate in the presence of protein resulted in the formation of antigenic materials suggested that polyunsaturated fatty acids are sources of acrolein that cause the production of protein-bound acrolein. These data suggest that the protein-bound acrolein represents potential markers of oxidative stress and long-term damage to protein in aging, atherosclerosis, and diabetes.
Alendronate mechanism of action: geranylgeraniol, an intermediate in the mevalonate pathway, prevents inhibition of osteoclast formation, bone resorption, and kinase activation
in vitro
Authors: Fisher J. E. , Rogers M. J. , Halasy J. M. , Luckman S. P. , Hughes D. E. , Masarachia P. J. , Wesolowski G. , Russell R. G. G. , Rodan G. A. , Reszka A. A.
Published: July 2002

Nitrogen-containing bisphosphonates were shown to cause macrophage apoptosis by inhibiting enzymes in the biosynthetic pathway leading from mevalonate to cholesterol. This study suggests that, in osteoclasts, geranylgeranyl diphosphate, the substrate for prenylation of most GTP binding proteins, is likely to be the crucial intermediate affected by these bisphosphonates. We report that murine osteoclast formation in culture is inhibited by both lovastatin, an inhibitor of hydroxymethylglutaryl CoA reductase, and alendronate. Lovastatin effects are blocked fully by mevalonate and less effectively by geranylgeraniol whereas alendronate effects are blocked partially by mevalonate and more effectively by geranylgeraniol. Alendronate inhibition of bone resorption in mouse calvaria also is blocked by mevalonate whereas clodronate inhibition is not. Furthermore, rabbit osteoclast formation and activity also are inhibited by lovastatin and alendronate. The lovastatin effects are prevented by mevalonate or geranylgeraniol, and alendronate effects are prevented by geranylgeraniol. Farnesol and squalene are without effect. Signaling studies show that lovastatin and alendronate activate in purified osteoclasts a 34-kDa kinase. Lovastatin-mediated activation is blocked by mevalonate and geranylgeraniol whereas alendronate activation is blocked by geranylgeraniol. Together, these findings support the hypothesis that alendronate, acting directly on osteoclasts, inhibits a rate-limiting step in the cholesterol biosynthesis pathway, essential for osteoclast function. This inhibition is prevented by exogenous geranylgeraniol, probably required for prenylation of GTP binding proteins that control cytoskeletal reorganization, vesicular fusion, and apoptosis, processes involved in osteoclast activation and survival.
Structural requirements of ligands for the oxysterol liver X receptors LXRα and LXRβ
Authors: Janowski Bethany A. , Grogan Michael J. , Jones Stacey A. , Wisely G. Bruce , Kliewer Steven A. , Corey Elias J. , Mangelsdorf David J.
Published: July 2002

LXRα and -β are nuclear receptors that regulate the metabolism of several important lipids, including cholesterol and bile acids. Previously, we have proposed that LXRs regulate these pathways through their interaction with specific, naturally occurring oxysterols, including 22(
R
)-hydroxycholesterol, 24(
S
)-hydroxycholesterol, and 24(
S
),25-epoxycholesterol. Using a ligand binding assay that incorporates scintillation proximity technology to circumvent many of the problems associated with assaying extremely hydrophobic ligands, we now demonstrate that these oxysterols bind directly to LXRs at concentrations that occur
in vivo
. To characterize further the structural determinants required for potent LXR ligands, we synthesized and tested a series of related compounds for binding to LXRs and activation of transcription. These studies revealed that position-specific monooxidation of the sterol side chain is requisite for LXR high-affinity binding and activation. Enhanced binding and activation can also be achieved through the use of 24-oxo ligands that act as hydrogen bond acceptors in the side chain. In addition, introduction of an oxygen on the sterol B-ring results in a ligand with LXRα-subtype selectivity. These results support the hypothesis that naturally occurring oxysterols are physiological ligands for LXRs and show that a rational, structure-based approach can be used to design potent LXR ligands for pharmacologic use.
Plant genetic resources: What can they contribute toward increased crop productivity?
Authors: Hoisington David , Khairallah Mireille , Reeves Timothy , Ribaut Jean-Marcel , Skovmand Bent , Taba Suketoshi , Warburton Marilyn
Published: July 2002

To feed a world population growing by up to 160 people per minute,
with >90% of them in developing countries, will require an
astonishing increase in food production. Forecasts call for wheat to
become the most important cereal in the world, with maize close behind;
together, these crops will account for ≈80% of developing
countries’ cereal import requirements. Access to a range of genetic
diversity is critical to the success of breeding programs. The global
effort to assemble, document, and utilize these resources is enormous,
and the genetic diversity in the collections is critical to the
world’s fight against hunger. The introgression of genes that reduced
plant height and increased disease and viral resistance in wheat
provided the foundation for the “Green Revolution” and
demonstrated the tremendous impact that genetic resources can have on
production. Wheat hybrids and synthetics may provide the yield
increases needed in the future. A wild relative of maize,
Tripsacum
, represents an untapped genetic resource for
abiotic and biotic stress resistance and for apomixis, a trait that
could provide developing world farmers access to hybrid technology.
Ownership of genetic resources and genes must be resolved to ensure
global access to these critical resources. The application of molecular
and genetic engineering technologies enhances the use of genetic
resources. The effective and complementary use of all of our
technological tools and resources will be required for meeting the
challenge posed by the world’s expanding demand for food.
A role for the light-dependent phosphorylation of visual arrestin
Authors: Alloway Paul G. , Dolph Patrick J.
Published: July 2002

Arrestins are regulatory proteins that participate in the termination of G protein-mediated signal transduction. The major arrestin in theDrosophilavisual system, Arrestin 2 (Arr2), is phosphorylated in a light-dependent manner by a Ca2+/calmodulin-dependent protein kinase and has been shown to be essential for the termination of the visual signaling cascadein vivo. Here, we report the isolation of nine alleles of theDrosophilaphotoreceptor cell-specificarr2gene. Flies carrying each of these alleles underwent light-dependent retinal degeneration and displayed electrophysiological defects typical of previously identified arrestin mutants, including an allele encoding a protein that lacks the major Ca2+/calmodulin-dependent protein kinase site. The phosphorylation mutant had very low levels of phosphorylation and lacked the light-dependent phosphorylation observed with wild-type Arr2. Interestingly, we found that the Arr2 phosphorylation mutant was still capable of binding to rhodopsin; however, it was unable to release from membranes once rhodopsin had converted back to its inactive form. This finding suggests that phosphorylation of arrestin is necessary for the release of arrestin from rhodopsin. We propose that the sequestering of arrestin to membranes is a possible mechanism for retinal disease associated with previously identified rhodopsin alleles in humans.
Selective activation of the versican promoter by epithelial– mesenchymal interactions during hair follicle development
Authors: Kishimoto Jiro , Ehama Ritsuko , Wu Lin , Jiang Shuwei , Jiang Nanyan , Burgeson Robert E.
Published: July 2002

Interaction between the epithelium and the mesenchyme is an essential feature of organogenesis, including hair follicle formation. The dermal papilla (DP), a dense aggregate of specialized dermis-derived stromal cells located at the bottom of the follicle, is a major component of hair that signals the follicular epithelial cells to prolong the hair growth process. However, little is known about DP-specific gene activation with regard to hair induction. In this study we demonstrate that a short fragment (839 bp) of the human versican (a core protein of one of the matrix chondroitin sulfate proteoglycans) promoter is sufficient to activate
lacZ
reporter gene expression in the DP of postnatal transgenic mice and also in the condensed mesenchyme (the origin of the DP) beneath the hair placode during hair follicle embryogenesis. Using the same versican promoter with green fluorescent protein (GFP), large numbers of fresh pelage DP cells were isolated from newborn transgenic skin by high-speed cell sorting. These GFP-positive DP cells showed abundant versican mRNA, confirming that the reporter molecules reflected endogenous versican gene expression. These sorted GFP-positive cells showed DP-like morphology in culture, but both GFP and versican expression was lost during primary culture.
In vivo
hair growth assays showed that GFP-positive cells could induce hair when grafted with epithelial cells, whereas GFP-negative cells grafted with epithelium or GFP-positive cells alone did not. These results suggest that versican may play an essential role both in mesenchymal condensation and in hair induction.
Behavioral reduction of infection risk
Authors: Kiesecker Joseph M. , Skelly David K. , Beard Karen H. , Preisser Evan
Published: July 2002

Evolutionary biologists have long postulated that there should be fitness advantages to animals that are able to recognize and avoid conspecifics infected with contact-transmitted disease. This avoidance hypothesis is in direct conflict with much of epidemiological theory, which is founded on the assumptions that the likelihood of infection is equal among members of a population and constant over space. The inconsistency between epidemiological theory and the avoidance hypothesis has received relatively little attention because, to date, there has been no evidence that animals can recognize and reduce infection risk from conspecifics. We investigated the effects of
Candida humicola
, a pathogen that reduces growth rates and can cause death of tadpoles, on associations between infected and uninfected individuals. Here we demonstrate that bullfrog (
Rana catesbeiana
) tadpoles avoid infected conspecifics because proximity influences infection. This avoidance behavior is stimulated by chemical cues from infected individuals and thus does not require direct contact between individuals. Such facultative modulations of disease infection risk may have critical consequences for the population dynamics of disease organisms and their impact on host populations.
Protective effects of estrogen in a rat model of age-related cataracts
Authors: Bigsby Robert M. , Cardenas Horacio , Caperell–Grant Andrea , Grubbs Clinton J.
Published: July 2002

Women have a higher incidence of cataracts, and epidemiologic data suggest that the increased risk may be caused by a lack of estrogen in postmenopausal years. We have examined the effects of estrogen on methylnitrosourea (MNU)-induced cataractogenesis in Sprague–Dawley rats. Animals were ovariectomized, injected with MNU, and treated with estradiol or estrone by a continuous-release, subcutaneous Silastic implant, or they received an empty Silastic implant (no hormone). In the no-hormone group, rats developed opaque lenses approximately 6 months after MNU treatment. By 8 months, 74% (14/19) of the no-hormone rats had evident opacity in one or both eyes by simple gross inspection; 58% (22/38) of the eyes in this group were opaque. Estradiol or estrone treatment reduced the incidence of cataractous eyes to 12% or 25%, respectively. Lenses were examined under a dissecting microscope for light transmission. The lenses of the group treated with no hormone had light transmission of 26% ± 9.2%, whereas lenses from the estradiol-treated animals had light transmission of 72% ± 5.8%. Histological examination revealed that the anterior cortices of the opaque lenses were disrupted and showed the hallmark signs of age-related cataracts; in addition, some eyes that appeared clear by macroscopic examination showed the early histologic signs of cataractogenesis. It was demonstrated with reverse transcription–PCR that lens cells express both α and β types of estrogen receptor, suggesting that the protective effects of the hormones may be a direct, receptor-mediated phenomenon. Thus, the MNU-treated, ovariectomized rat serves as a model for age-related cataractogenesis, and observation of a clear protective effect of estrogens in this system supports the implications of epidemiologic data.
Neurotrophins: Peripherally and centrally acting modulators of tactile stimulus-induced inflammatory pain hypersensitivity
Authors: Mannion R. J. , Costigan M. , Decosterd I. , Amaya F. , Ma Q.-P. , Holstege J. C. , Ji R.-R. , Acheson A. , Lindsay R. M. , Wilkinson G. A. , Woolf C. J.
Published: July 2002

Brain-derived neurotrophic factor (BDNF) is expressed in nociceptive sensory neurons and transported anterogradely to the dorsal horn of the spinal cord where it is located in dense core vesicles in C-fiber terminals. Peripheral inflammation substantially up-regulates BDNF mRNA and protein in the dorsal root ganglion (DRG) in a nerve growth factor-dependent fashion and results in novel expression of BDNF by DRG neurons with myelinated axons. C-fiber electrical activity also increases BDNF expression in the DRG, and both inflammation and activity increase full-length TrkB receptor levels in the dorsal horn. Sequestration of endogenous BDNF/neurotrophin 4 by intraspinal TrkB-Fc fusion protein administration does not, in noninflamed animals, change basal pain sensitivity nor the mechanical hypersensitivity induced by peripheral capsaicin administration, a measure of C fiber-mediated central sensitization. TrkB-Fc administration also does not modify basal inflammatory pain hypersensitivity, but does block the progressive hypersensitivity elicited by low-intensity tactile stimulation of inflamed tissue. BDNF, by virtue of its nerve growth factor regulation in sensory neurons including novel expression in A fibers, has a role as a central modulator of tactile stimulus-induced inflammatory pain hypersensitivity.
Behavioral economics: Reunifying psychology and economics
Authors: Camerer Colin
Published: July 2002

“Behavioral economics” improves the realism of the psychological assumptions underlying economic theory, promising to reunify psychology and economics in the process. Reunification should lead to better predictions about economic behavior and better policy prescriptions.
Involvement of specific calmodulin isoforms in salicylic acid-independent activation of plant disease resistance responses
Authors: Heo Won Do , Lee Sang Hyoung , Kim Min Chul , Kim Jong Cheol , Chung Woo Sik , Chun Hyun Jin , Lee Kyoung Joo , Park Chan Young , Park Hyeong Cheol , Choi Ji Young , Cho Moo Je
Published: July 2002

The Ca
2+
signal is essential for the activation of plant defense responses, but downstream components of the signaling pathway are still poorly defined. Here we demonstrate that specific calmodulin (CaM) isoforms are activated by infection or pathogen-derived elicitors and participate in Ca
2+
-mediated induction of plant disease resistance responses. Soybean CaM (SCaM)-4 and SCaM-5 genes, which encode for divergent CaM isoforms, were induced within 30 min by a fungal elicitor or pathogen, whereas other SCaM genes encoding highly conserved CaM isoforms did not show such response. This pathogen-triggered induction of these genes specifically depended on the increase of intracellular Ca
2+
level. Constitutive expression of SCaM-4 and SCaM-5 in transgenic tobacco plants triggered spontaneous induction of lesions and induces an array of systemic acquired resistance (SAR)-associated genes. Surprisingly, these transgenic plants have normal levels of endogenous salicylic acid (SA). Furthermore, coexpression of
nahG
gene did not block the induction of SAR-associated genes in these transgenic plants, indicating that SA is not involved in the SAR gene induction mediated by SCaM-4 or SCaM-5. The transgenic plants exhibit enhanced resistance to a wide spectrum of virulent and avirulent pathogens, including bacteria, fungi, and virus. These results suggest that specific CaM isoforms are components of a SA-independent signal transduction chain leading to disease resistance.
Reverse biochemistry: Use of macromolecular protease inhibitors to dissect complex biological processes and identify a membrane-type serine protease in epithelial cancer and normal tissue
Authors: Takeuchi Toshihiko , Shuman Marc A. , Craik Charles S.
Published: July 2002

Serine proteases of the chymotrypsin fold are of great
interest because they provide detailed understanding of their enzymatic
properties and their proposed role in a number of physiological and
pathological processes. We have been developing the macromolecular
inhibitor ecotin to be a “fold-specific” inhibitor that is
selective for members of the chymotrypsin-fold class of
proteases. Inhibition of protease activity through the use of wild-type
and engineered ecotins results in inhibition of rat prostate
differentiation and retardation of the growth of human PC-3 prostatic
cancer tumors. In an effort to identify the proteases that may be
involved in these processes, reverse transcription–PCR with PC-3
poly(A)+ mRNA was performed by using degenerate oligonucleotide
primers. These primers were designed by using conserved protein
sequences unique to chymotrypsin-fold serine proteases. Five
proteases were identified: urokinase-type plasminogen activator, factor
XII, protein C, trypsinogen IV, and a protease that we refer to as
membrane-type serine protease 1 (MT-SP1). The cloning and
characterization of the MT-SP1 cDNA shows that it encodes a mosaic
protein that contains a transmembrane signal anchor, two CUB domains,
four LDLR repeats, and a serine protease domain. Northern blotting
shows broad expression of MT-SP1 in a variety of epithelial tissues
with high levels of expression in the human gastrointestinal tract and
the prostate. A His-tagged fusion of the MT-SP1 protease domain was
expressed in
Escherichia coli
, purified, and
autoactivated. Ecotin and variant ecotins are subnanomolar inhibitors
of the MT-SP1 activated protease domain, suggesting a possible role for
MT-SP1 in prostate differentiation and the growth of prostatic
carcinomas.
Tissue concentration of heparin, not administered dose, correlates with the biological response of injured arteries
in vivo
Authors: Lovich Mark A. , Edelman Elazer R.
Published: July 2002

Drug activity is often studied in well controlled and characterized cellular environments
in vitro
. However, the biology of cells in culture is only a part of the tissue behavior
in vivo
. Quantitative studies of the dose response to drugs
in vivo
have been limited by the inability to reliably determine or predict the concentrations achieved in tissues. We developed a method to study the dose response of injured arteries to exogenous heparin
in vivo
by providing steady and predictable arterial levels of drug. Controlled-release devices were fabricated to direct heparin uniformly and at a steady rate to the adventitial surface of balloon-injured rat carotid arteries. We predicted the distribution of heparin throughout the arterial wall by using computational simulations of intravascular drug binding and transport, and we correlated these concentrations with the biologic response of the tissues. This allowed the estimation of the arterial concentration of heparin required to maximally inhibit intimal hyperplasia after injury
in vivo
, 0.3 mg/ml. This estimation of the required concentration of drug seen by a specific tissue is independent of the route of administration and holds for all forms of drug release. In this way we may now be able to evaluate the potential of widely disparate forms of drug release and to finally create some rigorous criteria by which to guide the development of particular delivery strategies for local diseases.
An endothelin receptor B antagonist inhibits growth and induces cell death in human melanoma cells
in vitro
and
in vivo
Authors: Lahav Ronit , Heffner Garrett , Patterson Paul H.
Published: July 2002

Activation of the endothelin receptor B (ETRB) in cultured melanocyte precursors promotes cell proliferation while inhibiting differentiation, two hallmarks of malignant transformation. We therefore tested whether ETRB has a similar role in malignant transformation of melanoma. When tested in culture, we find that the selective ETRB antagonist BQ788 can inhibit the growth of seven human melanoma cell lines, but not a human kidney cell line. This inhibition often is associated with increases in pigmentation and in the dendritic shape that is characteristic of mature melanocytes. In three cell lines we also observe a major increase in cell death. In contrast, the endothelin receptor A (ETRA) antagonist BQ123 does not have these effects, although all the cell lines express both ETRA and ETRB mRNA. Extending these studies
in vivo
, we find that administration of BQ788 significantly slows human melanoma tumor growth in nude mice, including a complete growth arrest in half of the mice treated systemically. Histological examination of tumor sections suggests that BQ788 also enhances melanoma cell death
in vivo
. Thus, ETRB inhibitors may be beneficial for the treatment of melanoma.
Detection of human neurotropic JC virus DNA sequence and expression of the viral oncogenic protein in pediatric medulloblastomas
Authors: Krynska Barbara , Del Valle Luis , Croul Sidney , Gordon Jennifer , Katsetos Christos D. , Carbone Michele , Giordano Antonio , Khalili Kamel
Published: July 2002

Medulloblastoma represents greater than 25% of childhood intracranial neoplasms and is considered a highly malignant tumor. This tumor, which arises predominantly in the cerebellar vermis, preferentially affects children between the ages of 5 and 15. Although the etiology of medulloblastomas in humans remains unknown, results from several experiments have indicated that the human neurotropic JC virus (JCV) is able to induce cerebellar neoplasms in rodents that exhibit a phenotype similar to that of human medulloblastomas. JCV is a polyomavirus that is widespread in the human population, with infection occurring most frequently in early childhood. In this study, we have examined the possible association of JCV with human medulloblastomas. By using PCR techniques we demonstrate that 11 of 23 samples of tumor tissue contain DNA sequences corresponding to three different regions of the JCV genome. More importantly, we demonstrate the presence of DNA sequences encoding the N- and C-terminal regions of the JCV oncogenic protein, T antigen, in 11 of 23 samples and the production of T antigen in the nuclei of 4 samples of tumor tissue. These observations provide evidence for a possible association of JCV with human medulloblastomas.
Specificity in transforming growth factor β-induced transcription of the plasminogen activator inhibitor-1 gene: Interactions of promoter DNA, transcription factor μE3, and Smad proteins
Authors: Hua Xianxin , Miller Zachary A. , Wu Geng , Shi Yigong , Lodish Harvey F.
Published: July 2002

Transforming growth factor β (TGF-β) regulates a broad range of biological processes, including cell growth, development, differentiation, and immunity. TGF-β signals through its cell surface receptor serine kinases that phosphorylate Smad2 or Smad3 proteins. Because Smad3 and its partner Smad4 bind to only 4-bp Smad binding elements (SBEs) in DNA, a central question is how specificity of TGF-β-induced transcription is achieved. We show that Smad3 selectively binds to two of the three SBEs in PE2.1, a TGF-β-inducible fragment of the plasminogen activator inhibitor-1 promoter, to mediate TGF-β-induced transcription; moreover, a precise 3-bp spacer between one SBE and the E-box, a binding site for transcription factor μE3 (TFE3), is essential for TGF-β-induced transcription. Whereas an isolated Smad3 MH1 domain binds to TFE3, TGF-β receptor-mediated phosphorylation of full-length Smad3 enhances its binding to TFE3. Together, these studies elucidate an important mechanism for specificity in TGF-β-induced transcription of the plasminogen activator inhibitor-1 gene.
Differentially expressed protein Pdcd4 inhibits tumor promoter-induced neoplastic transformation
Authors: Cmarik Joan L. , Min Hongzhong , Hegamyer Glenn , Zhan Shuning , Kulesz-Martin Molly , Yoshinaga Hidetoshi , Matsuhashi Sachiko , Colburn Nancy H.
Published: July 2002

An mRNA differential display comparison of mouse JB6 promotion-sensitive (P+) and -resistant (P−) cells identified a novel gene product that inhibits neoplastic transformation. The JB6 P+ and P− cells are genetic variants that differ in their transformation response to tumor promoters; P+ cells form anchorage-independent colonies that are tumorigenic, and P− cells do not. A differentially displayed fragment,A7-1, was preferentially expressed in P− cells at levels ≥10-fold those in P+ cells, making its mRNA a candidate inhibitor of neoplastic transformation. AnA7-1cDNA was isolated that was identical to murinePdcd4gene cDNAs, also known asMA-3orTIS, and analogous to humanH731and197/15a. Until now, the function of the Pdcd4 protein has been unknown. Paralleling the mRNA levels, Pdcd4 protein levels were greater in P− than in P+ cells.Pdcd4mRNA was also expressed at greater levels in the less progressed keratinocytes of another mouse skin neoplastic progression series. To test the hypothesis that Pdcd4 inhibits tumor promoter-induced transformation, stable cell lines expressing antisensePdcd4were generated from parental P− cells. The reduction of Pdcd4 proteins in antisense lines was accompanied by acquisition of a transformation-sensitive (P+) phenotype. The antisense-transfected cells were reverted to their initial P− phenotype by overexpression of aPdcd4sense fragment. These observations demonstrate that the Pdcd4 protein inhibits neoplastic transformation.
Photoassembly of the manganese cluster and oxygen evolution from monomeric and dimeric CP47 reaction center photosystem II complexes
Authors: Büchel Claudia , Barber Jim , Ananyev Gennady , Eshaghi Said , Watt Richard , Dismukes Charles
Published: July 2002

Isolated subcomplexes of photosystem II from spinach (CP47RC), composed of D1, D2, cytochrome b559, CP47, and a number of hydrophobic small subunits but devoid of CP43 and the extrinsic proteins of the oxygen-evolving complex, were shown to reconstitute the Mn4Ca1Clxcluster of the water-splitting system and to evolve oxygen. The photoactivation process in CP47RC dimers proceeds by the same two-step mechanism as observed in PSII membranes and exhibits the same stoichiometry for Mn2+, but with a 10-fold lower affinity for Ca2+and an increased susceptibility to photodamage. After the lower Ca2+affinity and the 10-fold smaller absorption cross-section for photons in CP47 dimers is taken into account, the intrinsic rate constant for the rate-limiting calcium-dependent dark step is indistinguishable for the two systems. The monomeric form of CP47RC also showed capacity to photoactivate and catalyze water oxidation, but with lower activity than the dimeric form and increased susceptibility to photodamage. After optimization of the various parameters affecting the photoactivation process in dimeric CP47RC subcores, 18% of the complexes were functionally reconstituted and the quantum efficiency for oxygen production by reactivated centers approached 96% of that observed for reconstituted photosystem II-enriched membranes.
Varying the number of telomere-bound proteins does not alter telomere length in
tel1Δ
cells
Authors: Ray Alo , Runge Kurt W.
Published: July 2002

Yeast telomere DNA consists of a continuous, ≈330-bp tract of the heterogeneous repeat TG
1-3
with irregularly spaced, high affinity sites for the protein Rap1p. Yeast monitor, or count, the number of telomeric Rap1p C termini in a negative feedback mechanism to modulate the length of the terminal TG
1-3
repeats, and synthetic telomeres that tether Rap1p molecules adjacent to the TG
1-3
tract cause wild-type cells to maintain a shorter TG
1-3
tract. To identify trans-acting proteins required to count Rap1p molecules, these same synthetic telomeres were placed in two short telomere mutants:
yku70Δ
(which lack the yeast Ku70 protein) and
tel1Δ
(which lack the yeast ortholog of ATM). Although both mutants maintain telomeres with ≈100 bp of TG
1-3
, only
yku70Δ
cells maintained shorter TG
1-3
repeats in response to internal Rap1p molecules. This distinct response to internal Rap1p molecules was not caused by a variation in Rap1p site density in the TG
1-3
repeats as sequencing of
tel1Δ
and
yku70Δ
telomeres showed that both strains have only five to six Rap1p sites per 100-bp telomere. In addition, the
tel1Δ
short telomere phenotype was epistatic to the unregulated telomere length caused by deletion of the Rap1p C-terminal domain. Thus, the length of the TG
1-3
repeats in
tel1Δ
cells was independent of the number of the Rap1p C termini at the telomere. These data indicate that
tel1Δ
cells use an alternative mechanism to regulate telomere length that is distinct from monitoring the number of telomere binding proteins.
Protein kinase C potentiation of
N
-methyl-
d
-aspartate receptor activity is not mediated by phosphorylation of
N
-methyl-
d
-aspartate receptor subunits
Authors: Zheng Xin , Zhang Ling , Wang Alice P. , Bennett Michael V. L. , Zukin R. Suzanne
Published: July 2002

N
-methyl-
d
-aspartate receptors (NMDARs) are Ca
2+
-permeable glutamate-gated ion channels whose physiological properties in neurons are modulated by protein kinase C (PKC). The present study was undertaken to determine the role in PKC-induced potentiation of the NR1 and NR2A C-terminal tails, which serve as targets of PKC phosphorylation [Tingley, W. G., Ehlers, M. D., Kameyama, K., Doherty, C., Ptak, J. B., Riley, C. T. & Huganir, R. L. (1997)
J. Biol. Chem.
272, 5157–5166]. Serine residue 890 in the C1 cassette is a primary target of PKC phosphorylation and a critical residue in receptor clustering at the membrane. We report herein that the presence of the C1 cassette reduces PKC potentiation and that mutation of Ser-890 significantly restores PKC potentiation. Splicing out or deletion of other C-terminal cassettes singly or in combination had little or no effect on PKC potentiation. Moreover, experiments involving truncation mutants reveal the unexpected finding that NMDARs assembled from subunits lacking all known sites of PKC phosphorylation can show PKC potentiation. These results indicate that PKC-induced potentiation of NMDAR activity does not occur by direct phosphorylation of the receptor protein but rather of associated targeting, anchoring, or signaling protein(s). PKC potentiation of NMDAR function is likely to be an important mode of NMDAR regulation
in vivo
and may play a role in NMDA-dependent long-term potentiation.
A binding site for homeodomain and Pax proteins is necessary for L1 cell adhesion molecule gene expression by Pax-6 and bone morphogenetic proteins
Authors: Meech Robyn , Kallunki Pekka , Edelman Gerald M. , Jones Frederick S.
Published: July 2002

The cell adhesion molecule L1 regulates axonal guidance and fasciculation during development. We previously identified the regulatory region of the L1 gene and showed that it was sufficient for establishing the neural pattern of L1 expression in transgenic mice. In the present study, we characterize a DNA element within this region called the HPD that contains binding motifs for both homeodomain and Pax proteins and responds to signals from bone morphogenetic proteins (BMPs). An ATTA sequence within the core of the HPD was required for binding to the homeodomain protein Barx2 while a separate paired domain recognition motif was necessary for binding to Pax-6. In cellular transfection experiments, L1-luciferase reporter constructs containing the HPD were activated an average of 4-fold by Pax-6 in N2A cells and 5-fold by BMP-2 and BMP-4 in Ng108 cells. Both of these responses were eliminated on deletion of the HPD from L1 constructs. In transgenic mice, deletion of the HPD from an
L1-lacZ
reporter resulted in a loss of β-galactosidase expression in the telencephalon and mesencephalon. Collectively, our experiments indicate that the HPD regulates L1 expression in neural tissues via homeodomain and Pax proteins and is likely to be a target of BMP signaling during development.
Troponin I is present in human cartilage and inhibits angiogenesis
Authors: Moses Marsha A. , Wiederschain Dmitri , Wu Inmin , Fernandez Cecilia A. , Ghazizadeh Vahid , Lane William S. , Flynn Evelyn , Sytkowski Arthur , Tao Terence , Langer Robert
Published: July 2002

Cartilage is an avascular and relatively tumor-resistant tissue. Work from a number of laboratories, including our own, has demonstrated that cartilage is an enriched source of endogenous inhibitors of angiogenesis. In the course of a study designed to identify novel cartilage-derived inhibitors of new capillary growth, we have purified an inhibitory protein that was identified by peptide microsequencing and protein database analysis as troponin I (TnI). TnI is a subunit of the troponin complex (troponin-C and troponin-T being the other two), which, along with tropomyosin, is responsible for the calcium-dependent regulation of striated muscle contraction; independently, TnI is capable of inhibiting actomyosin ATPase. Because troponin has never previously been reported to be present in cartilage, we have cloned and expressed the cDNA of human cartilage TnI, purified this protein to apparent homogeneity, and demonstrated that it is a potent and specific inhibitor of angiogenesis
in vivo
and
in vitro
, as well as of tumor metastasis
in vivo
.
Seasonal neuroplasticity in the songbird telencephalon: A role for melatonin
Authors: Bentley George E. , Van’t Hof Thomas J. , Ball Gregory F.
Published: July 2002

Neuroplasticity in the vocal control system of songbirds is strongly influenced by seasonal fluctuations in circulating testosterone. These seasonally plastic telencephalic structures are implicated in the learning and production of song in songbirds. The role of the indoleamine melatonin in seasonal adaptations in birds has remained unclear. In this experiment, European starlings were castrated to remove the neuromodulating activity of gonadal steroids and were exposed to different photoperiods to induce reproductive states characteristic of different seasonal conditions. Long days increased the volume of the song-control nucleus high vocal center compared with its volume on short days. Exogenous melatonin attenuated the long-day-induced volumetric increase in high vocal center and also decreased the volume of another song-control nucleus, area X. This effect was observed regardless of reproductive state. To our knowledge, this is the first direct evidence of a role for melatonin in functional plasticity within the central nervous system of vertebrates.
An empirical basis for Mach bands
Authors: Lotto R. Beau , Williams S. Mark , Purves Dale
Published: July 2002

Mach bands, the illusory brightness maxima and minima perceived at the initiation and termination of luminance gradients, respectively, are generally considered a direct perceptual manifestation of lateral inhibitory interactions among retinal or other lower order visual neurons. Here we examine an alternative explanation, namely that Mach bands arise as a consequence of real-world luminance gradients. In this first of two companion papers, we analyze the natural sources of luminance gradients, demonstrating that real-world gradients arising from curved surfaces are ordinarily adorned by photometric highlights and lowlights in the position of the illusory bands. The prevalence of such gradients provides an empirical basis for the generation of this perceptual phenomenon.
Circadian rhythms in
Neurospora crassa
: Lipid deficiencies restore robust rhythmicity to null
frequency
and
white-collar
mutants
Authors: Lakin-Thomas Patricia L. , Brody Stuart
Published: July 2002

The conidiation rhythm in the fungus
Neurospora crassa
is a model system for investigating the genetics of circadian clocks. Null mutants at the
frq (frequency)
locus (

frq
9

and

frq
10

) make no functional
frq
gene products and are arrhythmic under standard conditions. The
white-collar
strains (
wc-1
and
wc-2
) are insensitive to most effects of light, and are also arrhythmic. All three genes are proposed to be central components of the circadian oscillator. We have been investigating two mutants,
cel (chain-elongation)
and
chol-1 (choline-requirer)
, which are defective in lipid synthesis and affect the period and temperature compensation of the rhythm. We have constructed the double mutant strains

chol-1 frq
9

,

chol-1 frq
10

,
chol-1 wc-1
,

chol-1 wc-2, cel frq
9

, cel frq
10
, and
cel wc-2
. We find that these double mutant strains are robustly rhythmic when assayed under lipid-deficient conditions, indicating that free-running rhythmicity does not require the
frq, wc-1,
or
wc-2
gene products. The rhythms in the double mutant strains are similar to the
cel
and
chol-1
parents, except that they are less sensitive to light. This suggests that the
frq, wc-1,
and
wc-2
gene products may be components of a pathway that normally supplies input to a core oscillator to transduce light signals and sustain rhythmicity. This pathway can be bypassed when lipid metabolism is altered.
Design of a potent and selective inhibitor of the intermediate-conductance Ca2+-activated K+channel,IKCa1: A potential immunosuppressant
Authors: Wulff Heike , Miller Mark J. , Hänsel Wolfram , Grissmer Stephan , Cahalan Michael D. , Chandy K. George
Published: July 2002

The antimycotic clotrimazole, a potent inhibitor of the intermediate-conductance calcium-activated K+channel,IKCa1, is in clinical trials for the treatment of sickle cell disease and diarrhea and is effective in ameliorating the symptoms of rheumatoid arthritis. However, inhibition of cytochrome P450 enzymes by clotrimazole limits its therapeutic value. We have used a rational design strategy to develop a clotrimazole analog that selectively inhibitsIKCa1without blocking cytochrome P450 enzymes. A screen of 83 triarylmethanes revealed the pharmacophore for channel block to be different from that required for cytochrome P450 inhibition. The “IKCa1-pharmacophore” consists of a (2-halogenophenyl)diphenylmethane moiety substituted by an unsubstituted polar π-electron-rich heterocycle (pyrazole or tetrazole) or a −C
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N group, whereas cytochrome P450 inhibition absolutely requires the imidazole ring. A series of pyrazoles, acetonitriles, and tetrazoles were synthesized and found to selectively blockIKCa1. TRAM-34 (1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole) inhibits the cloned and the nativeIKCa1channel in human T lymphocytes with aKdof 20–25 nM and is 200- to 1,500-fold selective over other ion channels. Using TRAM-34, we show that blockingIKCa1in human lymphocytes, in the absence of P450-inhibition, results in suppression of mitogen-stimulated [3H]thymidine incorporation of preactivated lymphocytes with EC50-values of 100 nM-1 μM depending on the donor. Combinations of TRAM-34 and cyclosporin A are more effective in suppressing lymphocyte mitogenesis than either compound alone. Our studies suggest that TRAM-34 and related compounds may hold therapeutic promise as immunosuppressants.
Singular value decomposition for genome-wide expression data processing and modeling
Authors: Alter Orly , Brown Patrick O. , Botstein David
Published: July 2002

We describe the use of singular value decomposition in transforming genome-wide expression data from genes × arrays space to reduced diagonalized “eigengenes” × “eigenarrays” space, where the eigengenes (or eigenarrays) are unique orthonormal superpositions of the genes (or arrays). Normalizing the data by filtering out the eigengenes (and eigenarrays) that are inferred to represent noise or experimental artifacts enables meaningful comparison of the expression of different genes across different arrays in different experiments. Sorting the data according to the eigengenes and eigenarrays gives a global picture of the dynamics of gene expression, in which individual genes and arrays appear to be classified into groups of similar regulation and function, or similar cellular state and biological phenotype, respectively. After normalization and sorting, the significant eigengenes and eigenarrays can be associated with observed genome-wide effects of regulators, or with measured samples, in which these regulators are overactive or underactive, respectively.
Distinct cAMP response element-binding protein (CREB) domains stimulate different steps in a concerted mechanism of transcription activation
Authors: Kim Jeong-a , Lu Jingfang , Quinn Patrick G.
Published: July 2002

Hormones and neurotransmitters rapidly change patterns of gene
expression in target cells by activating protein kinases that
phosphorylate and modify the activity of CREB and other transcription
factors. Although CREB was initially characterized as mediating the
response to cAMP, CREB phosphorylation and activation are stimulated by
diverse extracellular signals and protein kinases in essentially all
cells and tissues. CREB stimulates transcription through a constitutive
activation domain (CAD), which interacts with the promoter recognition
factor TFIID, and through a kinase-inducible domain (KID), when Ser-133
is phosphorylated. The present study provides new insight into the
mechanism of activation by showing that each of the CREB domains
contributes to transcription initiation by stimulating sequential steps
in the transcription reaction. The CAD effectively assembled a
polymerase complex, as evidenced by constitutive activation
in
vivo
and stimulation of single-round transcription
in
vitro
. In contrast, phosphorylation of the KID in CREB
stimulated isomerization of the polymerase complex, as determined by
abortive initiation, and promoter clearance and/or reinitiation, as
measured by multiple rounds of transcription. Our results provide
evidence for a new model for CREB-mediated induction through a
concerted mechanism involving establishment of a polymerase complex by
the CAD, followed by stimulation of isomerization, promoter clearance,
and/or reinitiation by phosphorylated KID to enhance target gene
transcription.
Sensory neuron-specific sodium channel SNS is abnormally expressed in the brains of mice with experimental allergic encephalomyelitis and humans with multiple sclerosis
Authors: Black Joel A. , Dib-Hajj Sulayman , Baker David , Newcombe Jia , Cuzner M. Louise , Waxman Stephen G.
Published: July 2002

Clinical abnormalities in multiple sclerosis (MS) have classically
been considered to be caused by demyelination and/or axonal
degeneration; the possibility of molecular changes in neurons, such as
the deployment of abnormal repertoires of ion channels that would alter
neuronal electrogenic properties, has not been considered.
S
ensory
N
euron-
S
pecific
sodium channel SNS displays a depolarized voltage dependence, slower
activation and inactivation kinetics, and more rapid recovery from
inactivation than classical “fast” sodium channels. SNS is
selectively expressed in spinal sensory and trigeminal ganglion neurons
within the peripheral nervous system and is not expressed within the
normal brain. Here we show that sodium channel SNS mRNA and protein,
which are not present within the cerebellum of control mice, are
expressed within cerebellar Purkinje cells in a mouse model of MS,
chronic relapsing experimental allergic encephalomyelitis. We also
demonstrate SNS mRNA and protein expression within Purkinje cells from
tissue obtained postmortem from patients with MS, but not in control
subjects with no neurological disease. These results demonstrate a
change in sodium channel expression in neurons within the brain in an
animal model of MS and in humans with MS and suggest that abnormal
patterns of neuronal ion channel expression may contribute to clinical
abnormalities such as ataxia in these disorders.
Structural requirements of six naturally occurring isoforms of the IL-18 binding protein to inhibit IL-18
Authors: Kim Soo-Hyun , Eisenstein Miriam , Reznikov Leonid , Fantuzzi Giamila , Novick Daniela , Rubinstein Menachem , Dinarello Charles A.
Published: July 2002

A novel, constitutively expressed and secreted IL-18 binding protein (IL-18BP) neutralizes IL-18 and thereby suppresses the production of IFN-γ, resulting in reduced T-helper type 1 immune responses. In the present study, four human and two mouse isoforms, resulting from mRNA splicing and found in various cDNA libraries, were expressed, purified, and assessed for binding and neutralization of IL-18 biological activities. Human IL-18BP isoform a (IL-18BPa) exhibited the greatest affinity for IL-18 with a rapid on-rate, a slow off-rate, and a dissociation constant (Kd) of 399 pM. IL-18BPc shares the Ig domain of IL-18BPa except for the 29 C-terminal amino acids; theKdof IL-18BPc is 10-fold less (2.94 nM). Nevertheless, IL-18BPa and IL-18BPc neutralize IL-18 >95% at a molar excess of two. IL-18BPb and IL-18BPd isoforms lack a complete Ig domain and lack the ability to bind or neutralize IL-18. Murine IL-18BPc and IL-18BPd isoforms, possessing the identical Ig domain, also neutralize >95% murine IL-18 at a molar excess of two. However, murine IL-18BPd, which shares a common C-terminal motif with human IL-18BPa, also neutralizes human IL-18. Molecular modeling identified a large mixed electrostatic and hydrophobic binding site in the Ig domain of IL-18BP, which could account for its high affinity binding to the ligand. It is likely that preferential secretion of functional and nonfunctional isoforms of IL-18BP affect the immune response.
It's a bug's life
Authors: Patel Nipam H.
Published: July 2002

Although the mechanisms of
anterior-posterior axis formation
are well understood in
Drosophila
, both
embryological and molecular
studies suggest significant variation in the mechanisms
generating this axis within the Insecta class as a whole.