bluuebunny/crossref_metadata_2025 huggingface dataset:
Face encoding and recognition in the human brain.
Authors: Haxby J V , Ungerleider L G , Horwitz B , Maisog J M , Rapoport S I , Grady C L
Published: July 2002

A dissociation between human neural systems that participate in the encoding and later recognition of new memories for faces was demonstrated by measuring memory task-related changes in regional cerebral blood flow with positron emission tomography. There was almost no overlap between the brain structures associated with these memory functions. A region in the right hippocampus and adjacent cortex was activated during memory encoding but not during recognition. The most striking finding in neocortex was the lateralization of prefrontal participation. Encoding activated left prefrontal cortex, whereas recognition activated right prefrontal cortex. These results indicate that the hippocampus and adjacent cortex participate in memory function primarily at the time of new memory encoding. Moreover, face recognition is not mediated simply by recapitulation of operations performed at the time of encoding but, rather, involves anatomically dissociable operations.
Phosphorylation of MCM4 by cdc2 protein kinase inhibits the activity of the minichromosome maintenance complex.
Authors: Hendrickson M , Madine M , Dalton S , Gautier J
Published: July 2002

In eukaryotes, tight regulatory mechanisms ensure the ordered progression through the cell cycle phases. The mechanisms that prevent chromosomal DNA replication from taking place more than once each cell cycle are thought to involve the function of proteins of the minichromosome maintenance (MCM) family. Here, we demonstrate that Xenopus MCM4, a member of the MCM protein family related to Spcdc21/ ScCDC54, is part of a large protein complex comprising several other MCM proteins. MCM4 undergoes cell cycle-dependent phosphorylation both in cleaving embryos and in cell-free extracts. MCM4 phosphorylation starts concomitantly with the clearing of the MCM complex from the chromatin during S phase. Phosphorylation is carried out by cdc2/cyclinB protein kinase, which phosphorylates MCM4 in vitro at identical sites as the ones phosphorylated in vivo. Phosphorylation is specific for cdc2 protein kinase since MCM4 is not a substrate for other members of the cdk family. Furthermore, phosphorylation of MCM4 dramatically reduces its affinity for the chromatin. We propose that the cell cycle-dependent phosphorylation of MCM4 is a mechanism which inactivates the MCM complex from late S phase through mitosis, thus preventing illegitimate DNA replication during that period of the cell cycle.
A modular misexpression screen in Drosophila detecting tissue-specific phenotypes.
Authors: Rørth P
Published: July 2002

Genetic screens in Drosophila have lead to the discovery of many genes important for patterning and signal transduction in diverse organisms. Traditionally, the phenotypic effects of loss-of-function mutations are analyzed. As an alternative way to link genes and function, I have developed a versatile misexpression screen in Drosophila, the first such screen in higher eukaryotes. The screen identifies genes that, when over- or misexpressed in a pattern of interest, give a specific phenotype or modulate an existing mutant phenotype. It is based on Gal4 transactivation of a mobile enhancer and promoter that "targets" random endogenous genes for expression. The modular design of the screen allows directed expression in any temporal or spatial pattern. When activated in the developing eye, 4% of target inserts gave dominant phenotypes. One insertion was in the gene encoding Ras GTPase-activating protein; its overexpression phenotype was strongly enhanced by a mutation in Ras1. Thus, biologically relevant phenotypes and genetic interactions are identified using this method. The screen is a powerful new tool for developmental genetics; similar approaches can also be developed for other organisms.
Role for the class A macrophage scavenger receptor in the phagocytosis of apoptotic thymocytes in vitro.
Authors: Platt N , Suzuki H , Kurihara Y , Kodama T , Gordon S
Published: July 2002

Numerous immature thymocytes undergo apoptosis and are rapidly engulfed by phagocytic thymic macrophages. The macrophage surface receptors involved in apoptotic thymocyte recognition are unknown. We have examined the role of the class A macrophage scavenger receptor (SR-A) in the engulfment of apoptotic thymocytes. Uptake of steroid-treated apoptotic thymocytes by thymic and inflammatory-elicited SR-A positive macrophages is partially inhibited by an anti-SR-A mAb and more completely by a range of scavenger receptor ligands. Thymic macrophages from mice with targeted disruption of the SR-A gene show a 50% reduction in phagocytosis of apoptotic thymocytes in vitro. These data suggest that SR-A may play a role in the clearance of dying cells in the thymus.
Polyunsaturated fatty acids modulate sodium and calcium currents in CA1 neurons.
Authors: Vreugdenhil M , Bruehl C , Voskuyl R A , Kang J X , Leaf A , Wadman W J
Published: July 2002

Recent evidence indicates that long-chain polyunsaturated fatty acids (PUFAs) can prevent cardiac arrhythmias by a reduction of cardiomyocyte excitability. This was shown to be due to a modulation of the voltage-dependent inactivation of both sodium (INa) and calcium (ICa) currents. To establish whether PUFAs also regulate neuronal excitability, the effects of PUFAs on INa and ICa were assessed in CA1 neurons freshly isolated from the rat hippocampus. Extracellular application of PUFAs produced a concentration-dependent shift of the voltage dependence of inactivation of both INa and ICa to more hyperpolarized potentials. Consequently, they accelerated the inactivation and retarded the recovery from inactivation. The EC50 for the shift of the INa steady-state inactivation curve was 2.1 +/- 0.4 microM for docosahexaenoic acid (DHA) and 4 +/- 0.4 microM for eicosapentaenoic acid (EPA). The EC50 for the shift on the ICa inactivation curve was 2.1 +/- 0.4 for DHA and > 15 microM for EPA. Additionally, DHA and EPA suppressed both INa and ICa amplitude at concentrations > 10 microM. PUFAs did not affect the voltage dependence of activation. The monounsaturated oleic acid and the saturated palmitic acid were virtually ineffective. The combined effects of the PUFAs on INa and ICa may reduce neuronal excitability and may exert anticonvulsive effects in vivo.
A general method for determining helix packing in membrane proteinsin situ: Helices I and II are close to helix VII in the lactose permease ofEscherichia coli
Authors: Wu Jianhua , Kaback H. Ronald
Published: July 2002

It was previously shown that coexpression of the lactose permease ofEscherichia coliin two contiguous fragments leads to functional complementation. We demonstrate here that site-directed thiol crosslinking of coexpressed permease fragments can be used to determine helix proximityin situwithout the necessity of purifying the permease. After coexpression of the six N-terminal (N6) and six C-terminal (C6) transmembrane helices, each with a single Cys residue, crosslinking was carried out in native membranes and assessed by the mobility of anti-C-terminal-reactive polypeptides on immunoblots. A Cys residue at position 242 or 245 (helix VII) forms a disulfide with a Cys residue at either position 28 or 29 (helix I), but not with a Cys residue at position 27, which is on the opposite face of helix I, thereby indicating that the face of helix I containing Pro-28 and Phe-29 is close to helix VII. Similarly, a Cys residue at position 242 or 245 (helix VII) forms a disulfide with a Cys residue at either position 52 or 53 (helix II), but not with a Cys residue at position 54. Furthermore, low-efficiency crosslinking is observed between a Cys residue at position 52 or 53 and a Cys residue at position 361 (helix XI). The results indicate that helix VII lies in close proximity to both helices I and II and that helix II is also close to helix XI. The method should be applicable to a number of different polytopic membrane proteins.
Transformation of Plasmodium falciparum malaria parasites by homologous integration of plasmids that confer resistance to pyrimethamine.
Authors: Wu Y , Kirkman L A , Wellems T E
Published: July 2002

Plasmodium falciparum malaria parasites were transformed with plasmids containing P. falciparum or Toxoplasma gondii dihydrofolate reductase-thymidylate synthase (dhfr-ts) coding sequences that confer resistance to pyrimethamine. Under pyrimethamine pressure, transformed parasites were obtained that maintained the transfected plasmids as unrearranged episomes for several weeks. These parasite populations were replaced after 2 to 3 months by parasites that had incorporated the transfected DNA into nuclear chromosomes. Depending upon the particular construct used for transformation, homologous integration was detected in the P. falciparum dhfr-ts locus (chromosome 4) or in hrp3 and hrp2 sequences that were used in the plasmid constructs as gene control regions (chromosomes 13 and 8, respectively). Transformation by homologous integration sets the stage for targeted gene alterations and knock-outs that will advance understanding of P. falciparum.
Antigenic variation and the within-host dynamics of parasites.
Authors: Antia R , Nowak M A , Anderson R M
Published: July 2002

Many parasites exhibit antigenic variation within their hosts. We use mathematical models to investigate the dynamical interaction between an antigenically varying parasite and the host's immune system. The models incorporate antigenic variation in the parasite population and the generation of immune responses directed against (i) antigens specific to individual parasite variants and (ii) antigens common to all the parasite variants. Analysis of the models allows us to evaluate the relative importance of variant-specific and cross-reactive immune responses in controlling the parasite. Early in the course of infection within the host, when parasite diversity is below a defined threshold value (the value is determined by the biological properties of the parasite and of the host's immune response), the variant-specific immune responses are predominant. Later, when the parasite diversity is high, the cross-reactive immune response is largely responsible for controlling the parasitemia. It is argued that increasing antigenic diversity leads to a switch from variant-specific to cross-reactive immune responses. These simple models mimic various features of observed infections recorded in the experimental literature, including an initial peak in parasitemia, a long and variable duration of infection with fluctuating parasitemia that ends with either the clearance of the parasite or persistent infection.
Motor learning by field approximation.
Authors: Gandolfo F , Mussa-Ivaldi F A , Bizzi E
Published: July 2002

We investigated how human subjects adapt to forces perturbing the motion of their ams. We found that this kind of learning is based on the capacity of the central nervous system (CNS) to predict and therefore to cancel externally applied perturbing forces. Our experimental results indicate: (i) that the ability of the CNS to compensate for the perturbing forces is restricted to those spatial locations where the perturbations have been experienced by the moving arm. The subjects also are able to compensate for forces experienced at neighboring workspace locations. However, adaptation decays smoothly and quickly with distance from the locations where disturbances had been sensed by the moving limb. (ii) Our experiments also how that the CNS builds an internal model of the external perturbing forces in intrinsic (muscles and / or joints) coordinates.
BRCA1 is a component of the RNA polymerase II holoenzyme
Authors: Scully Ralph , Anderson Stephen F. , Chao David M. , Wei Wanjiang , Ye Liyan , Young Richard A. , Livingston David M. , Parvin Jeffrey D.
Published: July 2002

The familial breast–ovarian tumor suppressor gene product BRCA1 was found to be a component of the RNA polymerase II holoenzyme by several criteria. BRCA1 was found to copurify with the holoenzyme over multiple chromatographic steps. Other tested transcription activators that could potentially contact the holoenzyme were not stably associated with the holoenzyme as determined by copurification. Antibody specific for the holoenzyme component hSRB7 specifically purifies BRCA1. Immunopurification of BRCA1 complexes also specifically purifies transcriptionally active RNA polymerase II and transcription factors TFIIF, TFIIE, and TFIIH. Moreover, a BRCA1 domain, which is deleted in about 90% of clinically relevant mutations, participates in binding to the holoenzyme complex in cells. These data are consistent with recent data identifying transcription activation domains in the BRCA1 protein and link the BRCA1 tumor suppressor protein with the transcription process as a holoenzyme-bound protein.
Characteristic temperatures of folding of a small peptide
Authors: Hansmann Ulrich H. E. , Masuya Masato , Okamoto Yuko
Published: July 2002

We perform a generalized-ensemble simulation of a small peptide taking the interactions among all atoms into account. From this simulation we obtain thermodynamic quantities over a wide range of temperatures. In particular, we show that the folding of a small peptide is a multistage process associated with two characteristic temperatures, the collapse temperature
T
θ
and the folding temperature
T
ƒ
. Our results give supporting evidence for the energy landscape picture and funnel concept. These ideas were previously developed in the context of studies of simplified protein models, and here are checked in an all-atom Monte Carlo simulation.
Sequence-specific recognition of a subgenomic RNA promoter by a viral RNA polymerase
Authors: Siegel Robert W. , Adkins Scott , Kao C. Cheng
Published: July 2002

RNA templates of 33 nucleotides containing the brome mosaic virus (BMV) core subgenomic promoter were used to determine the promoter elements recognized by the BMV RNA-dependent RNA polymerase (RdRp) to initiate RNA synthesis. Nucleotides at positions −17, −14, −13, and −11 relative to the subgenomic initiation site must be maintained for interaction with the RdRp. Changes to every other nucleotide at these four positions allow predictions for the base-specific functional groups required for RdRp recognition. RdRp contact of the nucleotide at position −17 was suggested with a template competition assay. Comparison of the BMV subgenomic promoter to those from other plant and animal alphaviruses shows a remarkable degree of conservation of the nucleotides required for BMV subgenomic RNA synthesis. We show that the RdRp of the plant-infecting BMV is capable of accurately, albeit inefficiently, initiating RNA synthesis from the subgenomic promoter of the animal-infecting Semliki Forest virus. The sequence-specific recognition of RNA by the BMV RdRp is analogous to the recognition of DNA promoters by DNA-dependent RNA polymerases.
Participation of the nuclear cap binding complex in pre-mRNA 3′ processing
Authors: Flaherty Sean M. , Fortes Puri , Izaurralde Elisa , Mattaj Iain W. , Gilmartin Gregory M.
Published: July 2002

Communication between the 5′ and 3′ ends is a common feature of several aspects of eukaryotic mRNA metabolism. In the nucleus, the pre-mRNA 5′ end is bound by the nuclear cap binding complex (CBC). This RNA–protein complex plays an active role in both splicing and RNA export. We provide evidence for participation of CBC in the processing of the 3′ end of the message. Depletion of CBC from HeLa cell nuclear extract strongly reduced the endonucleolytic cleavage step of the cleavage and polyadenylation process. Cleavage was restored by addition of recombinant CBC. CBC depletion was found to reduce the stability of poly(A) site cleavage complexes formed in nuclear extract. We also provide evidence that the communication between the 5′ and 3′ ends of the pre-mRNA during processing is mediated by the physical association of the CBC/cap complex with 3′ processing factors bound at the poly(A) site. These observations, along with previous data on the function of CBC in splicing, illustrate the key role played by CBC in pre-mRNA recognition and processing. The data provides further support for the hypothesis that pre-mRNAs and mRNAs may exist and be functional in the form of “closed-loops,” due to interactions between factors bound at their 5′ and 3′ ends.
Prolonged production of NADPH oxidase-corrected granulocytes after gene therapy of chronic granulomatous disease
Authors: Malech Harry L. , Maples Phillip B. , Whiting-Theobald Narda , Linton Gilda F. , Sekhsaria Sudhir , Vowells Sarah J. , Li Fei , Miller Judi A. , DeCarlo Ellen , Holland Steven M. , Leitman Susan F. , Carter Charles S. , Butz Robert E. , Read Elizabeth J. , Fleisher Thomas A. , Schneiderman Richard D. , Van Epps Dennis E. , Spratt S. Kaye , Maack Christopher A. , Rokovich Joseph A. , Cohen Lawrence K. , Gallin John I.
Published: July 2002

Little is known about the potential for engraftment of autologous hematopoietic stem cells in human adults not subjected to myeloablative conditioning regimens. Five adult patients with the p47
phox
deficiency form of chronic granulomatous disease received intravenous infusions of autologous CD34
+
peripheral blood stem cells (PBSCs) that had been transduced
ex vivo
with a recombinant retrovirus encoding normal p47
phox
. Although marrow conditioning was not given, functionally corrected granulocytes were detectable in peripheral blood of all five patients. Peak correction occurred 3–6 weeks after infusion and ranged from 0.004 to 0.05% of total peripheral blood granulocytes. Corrected cells were detectable for as long as 6 months after infusion in some individuals. Thus, prolonged engraftment of autologous PBSCs and continued expression of the transduced gene can occur in adults without conditioning. This trial also piloted the use of animal protein-free medium and a blood-bank-compatible closed system of gas-permeable plastic containers for culture and transduction of the PBSCs. These features enhance the safety of PBSCs directed gene therapy.
The glyoxysomal and plastid molecular chaperones (70-kDa heat shock protein) of watermelon cotyledons are encoded by a single gene
Authors: Wimmer Bernhard , Lottspeich Friedrich , van der Klei Ida , Veenhuis Marten , Gietl Christine
Published: July 2002

The monoclonal a-70-kDa heat shock protein (hsp70) antibody recognizes in crude extracts from watermelon (
Citrullus vulgaris
) cotyledons two hsps with molecular masses of 70 and 72 kDa. Immunocytochemistry on watermelon cotyledon tissue and on isolated glyoxysomes identified hsp70s in the matrix of glyoxysomes and plastids. Affinity purification and partial amino acid determination revealed the 70-kDa protein to share high sequence identity with cytosolic hsp70s from a number of plant species, while the 72 kDa protein was very similar to plastid hsp70s from pea and cucumber. A full-length cDNA clone encoding the 72-kDa hsp70 was isolated and identified two start methionines in frame within the N-terminal presequence leading either to an N-terminal extension of 67 amino acids or to a shorter one of 47 amino acids. The longer presequence was necessary and sufficient to target a reporter protein into watermelon proplastids
in vitro
. The shorter extension starting from the second methionine within the long version harbored a consensus peroxisomal targeting signal (RT-X
5
-KL) that directed
in vivo
a reporter protein into peroxisomes of the yeast
Hansenula polymorpha
. Peroxisomal targeting was however prevented, when the 67-residue presequence was fused to the reporter protein, indicating that the peroxisomal targeting signal 2 information is hidden in this context. We propose that the 72-kDa hsp70 is encoded by a single gene, but targeted alternatively into two organelles by the modulated use of its presequence.
Evolutionary instability of the major histocompatibility complex class I loci in New World primates
Authors: Cadavid Luis F. , Shufflebotham Clare , Ruiz Francisco J. , Yeager Meredith , Hughes Austin L. , Watkins David I.
Published: July 2002

Homologues of the human major histocompatibility complex (MHC)HLA-A,-B,-E,-F, and-Gloci are present in all the Catarrhini (Old World primates, apes, and humans), and some of their allelic lineages have survived several speciation events. Analysis of 26 MHC class I cDNAs from seven different genera of New World primates revealed that the Callitrichinae (tamarins and marmosets) are an exception to these rules of MHC stability. In gene trees of primate MHC class I genes, sequences from the Callitrichinae cluster in a genus-specific fashion, whereas in the other genera of New World primates, as in the Catarrhini, they cluster in a transgeneric way. The genus-specific clustering of the Callitrichinae cDNAs indicates that there is no orthology between MHC class I loci in genera of this phyletic group. Additionally, the Callitrichinae genera exhibit limited variability of their MHC class I genes, in contrast to the high variability displayed by all other primates. Each Callitrichinae genus, therefore, expresses its own set of MHC class I genes, suggesting that an unusually high rate of turnover of loci occurs in this subfamily. The limited variability of MHC class I genes in the Callitrichinae is likely the result of the recent origin of these loci.
Antigen presentation in retroviral vector-mediated gene transfer in vivo
Authors: Song Elizabeth S. , Lee Virginia , Surh Charles D. , Lynn Andrea , Brumm Duane , Jolly Douglas J. , Warner John F. , Chada Sunil
Published: July 2002

We have examined mechanisms involved in gene transfer, protein expression, and antigen presentation after direct administration of retroviral vectors using a variety of antigen systems. We have identified transduced infiltrating cells at the injection site, and the majority of the infiltrating cells were of the monocyte/macrophage lineage. We found that the splenic dendritic cell fraction contained proviral DNA, expressed antigenic proteins, and was able to present antigens efficiently to the immune system. Furthermore, the dendritic cell fractions from retroviral vector-immunized mice were able to prime naive T cellsin vitro, and adoptive transfer ofin vitro-transduced dendritic cell fractions elicited antigen-specific cytotoxic T lymphocytes. These data suggest a role for dendritic cells in induction of immune responses elicited by retroviral vector-mediated gene transfer.
Regulation of sodium channel gene expression by class I antiarrhythmic drugs and n − 3 polyunsaturated fatty acids in cultured neonatal rat cardiac myocytes
Authors: Kang Jing X. , Li Yunyuan , Leaf Alexander
Published: July 2002

Previous studies have shown that chronic administration of class I antiarrhythmic drugs, which have definite inhibitory action on the fast Na
+
channel, result in up-regulation of cardiac Na
+
channel expression, and suggest that this effect may contribute to their deleterious effects during chronic administration. Recent studies have shown that the antiarrhythmic effects of free n − 3 polyunsaturated fatty acids (PUFA) are associated with an inhibition of the Na
+
channel. Whether the PUFA when used chronically will mimic the effect of the class I drugs on the expression of the Na
+
channel is not known. To answer this question, we determined the level of mRNA encoding cardiac Na
+
channels and the number of the Na
+
channels per cell in cultured neonatal rat cardiac myocytes after supplementation of the cells with the n − 3 PUFA eicosapentaenoic acid (EPA), the class I drug mexiletine, or both EPA and mexiletine for 3–4 days. The number of sodium channels was assessed with a radioligand binding assay using the sodium channel-specific toxin [
3
H]batrachotoxinin benzoate ([
3
H]BTXB). The supplementation of myocytes with mexiletine (20 μM) induced a 4-fold increase in [
3
H]BTXB specific binding to the cells. In contrast, chronic treatment with EPA (20 μM) alone did not significantly affect [
3
H]BTXB binding. However, the combination of EPA with mexiletine produced a 40–50% reduction in the [
3
H]BTXB binding, compared with that seen with mexiletine alone. RNA isolated from cardiac myocytes was probed with a 2.5-kb cRNA transcribed with T7 RNA polymerase from the clone Na-8.4, which encodes nucleotides 3361–5868 of the α-subunit of the R
IIA
sodium channel subtype. The changes in the level of mRNA encoding sodium channel α-subunit were correlated with comparable changes in sodium channel number in the cultured myocytes, indicating that regulation of transcription of mRNA or its processing and stability is primarily responsible for the regulation of sodium channel number. These data demonstrate that chronic EPA treatment not only does not up-regulate the cardiac sodium channel expression but also reduces the mexiletine-induced increase in the cardiac sodium channel expression.
The basic helix–loop–helix transcription factor BETA2/NeuroD is expressed in mammalian enteroendocrine cells and activates secretin gene expression
Authors: Mutoh Hiroyuki , Fung Brenda P. , Naya Francisco J. , Tsai Ming-Jer , Nishitani Junko , Leiter Andrew B.
Published: July 2002

The gene encoding the hormone secretin is expressed only in enteroendocrine S cells and insulin-producing pancreatic β cells during development. A 120-bp enhancer directs cell-specific expression of the rat secretin gene in secretin-expressing cells. The enhancer includes an E-box sequence, CAGCTG, which is important for transcriptional activity. To further characterize the role of the E box, a consensus binding site for basic helix–loop–helix (bHLH) proteins, we have examined factors that interact with this element in the secretin gene. The results suggest that transcription is activated by a recently isolated tissue-specific bHLH protein, BETA2, heterodimerized to the ubiquitously expressed bHLH proteins, Pan 1 and Pan 2, the rodent homologues of E47 and E12. The importance of BETA2 for transcriptional activation of secretin is further illustrated by antisense experiments inhibiting BETA2 expression in secretin-producing cell lines, which resulted in the inhibition of most E box-dependent transcription. Expression of BETA2 in a nonendocrine cell line conferred the ability to express secretin-reporter genes that are transcribed at minimal levels in the absence of BETA2. Secretin-producing enteroendocrine cells in the murine small intestine showed specific immunostaining with BETA2 antibodies, corroborating observations in cell lines. Thus BETA2 is to our knowledge the first transcription factor identified that specifically activates cell type-specific expression of an intestinal hormone gene.
Suppression of voltage-gated L-type Ca
2+
currents by polyunsaturated fatty acids in adult and neonatal rat ventricular myocytes
Authors: Xiao Yong-Fu , Gomez Ana Maria , Morgan James P. , Lederer W. J. , Leaf Alexander
Published: July 2002

Our recent data show that in cardiac myocytes polyunsaturated fatty acids (PUFAs) are antiarrhythmic. They reduce
I
Na
, shorten the action potential, shift the threshold for excitation to more positive potentials, and prolong the relative refractory period. In this study we use patch-clamp techniques in whole-cell mode and confocal Ca
2+
imaging to examine the effects of PUFAs on the voltage-gated L-type Ca
2+
current (
I
Ca,L
), elementary sarcoplasmic reticulum Ca
2+
-release events (Ca
2+
-sparks), and [Ca
2+
]
i
transients in isolated rat ventricular myocytes. Extracellular application of eicosapentaenoic acid (EPA; C20:5
n
− 3) produced a prompt and reversible concentration-dependent suppression of
I
Ca,L
. The concentration of EPA to produce 50% inhibition of
I
Ca
was 0.8 μM in neonatal rat heart cells and 2.1 μM in adult ventricular myocytes. While the EPA induced suppression of
I
Ca,L
, it did not significantly alter the shape of the current–voltage relation but did produce a small, but significant, negative shift of the steady-state inactivation curve. The inhibition of
I
Ca,L
was voltage- and time-dependent, but not use- or frequency-dependent. Other PUFAs, such as docosahexaenoic acid, arachidonic acid, linolenic acid, linoleic acid, conjugated linoleic acid, and eicosatetraynoic acid had similar effects on
I
Ca,L
as EPA. All-
trans
-retinoic acid, which had been shown to suppress induced arrhythmogenic activity in rat heart cells, also produced a significant inhibition of
I
Ca,L
. The saturated stearic acid and the monounsaturated oleic acid had no effect on
I
Ca,L
. Because both
I
Ca,L
and sarcoplasmic reticulum Ca
2+
-release underlie many cardiac arrhythmias, we examined the effects of EPA on
I
Ca,L
and Ca
2+
-sparks. While EPA suppressed both, it did not change the temporal or spatial character of the Ca
2+
-sparks, nor did it alter the ability of
I
Ca,L
to trigger Ca
2+
-sparks. We conclude that PUFAs may act as antiarrhythmic agents
in vivo
in normal and Ca
2+
-overloaded cells principally because they reduce Ca
2+
entry by blocking
I
Ca,L
. Furthermore, PUFAs act directly to decrease
I
Na
and
I
Ca,L
, but indirectly to reduce the [Ca
2+
]
i
transients and [Ca
2+
]
i
-activated membrane current. Although a negative inotropic action is associated with application of PUFAs, it is clear that by reducing
I
Ca,L
,
I
Na
and Ca
2+
-sparks, PUFAs can reduce spontaneous extrasystoles in the heart. The mechanisms by which PUFAs act are discussed.
GATA-1 transcription is controlled by distinct regulatory mechanisms during primitive and definitive erythropoiesis
Authors: Onodera Ko , Takahashi Satoru , Nishimura Shigeko , Ohta Jun , Motohashi Hozumi , Yomogida Kentaro , Hayashi Norio , Engel James Douglas , Yamamoto Masayuki
Published: July 2002

Transcription factor GATA-1 is required for the terminal differentiation of both the primitive and definitive erythroid cell lineages, and yet the regulatory mechanisms of GATA-1 itself are not well understood. To clarify how the GATA-1 gene is transcriptionally controlled
in vivo
, presumptive regulatory regions of the gene were tested by fusion to a reporter gene and then examined in transgenic mice. We found that a transcriptional control element located between −3.9 and −2.6 kb 5′ to the erythroid first exon serves as an activating element and that this sequence alone is sufficient to recapitulate the expression of GATA-1 (but uniquely in primitive erythroid cells). Addition of sequences from the GATA-1 first intron to this upstream element provides a necessary and sufficient condition for complete recapitulation of GATA-1 expression in both primitive and definitive erythroid cells. The first intron element does not possess intrinsic transcriptional activation potential when linked to the GATA-1 gene promoter but rather requires the upstream activating element for its activity. These experiments show that GATA-1 gene expression is regulated by discrete transcriptional control elements during definitive and primitive erythropoiesis: The 5′ element displays properties anticipated for a primitive erythroid cell-specific activating element, and the novel element within the GATA-1 first intron specifically augments this activity in definitive erythroid cells.
Synergism between hypersensitive sites confers long-range gene activation by the β-globin locus control region
Authors: Bresnick Emery H. , Tze Lina
Published: July 2002

The human β-globin locus control region (LCR) consists of four erythroid-specific DNaseI hypersensitive sites (HSs) at the 5′ end of the β-globin cluster. The LCR functions over a long distance on chromosome 11 to regulate transcription and replication of the β-globin genes. To determine whether the HSs function independently or as an integrated unit, we analyzed the requirements for long-range transcriptional activation. If the HSs function independently, individual HSs would be expected to have long-range activity. In contrast, if long-range activity requires multiple HSs, individual HSs would have a limited functional distance. HS2, HS3, and a miniLCR containing multiple HSs, were separated from a γ-globin promoter by fragments of phage λ DNA. After stable transfection into K562 cells, HS2 had strong enhancer activity, but only when positioned close to the promoter. HS3 also had strong enhancer activity, although it was weaker than HS2 and more sensitive to the spacer DNA. The miniLCR had the strongest enhancer activity and functioned even at a distance of 7.3 kb. A model is proposed in which synergistic interactions between HSs confer long-range activation by creating a
stable
LCR nucleoprotein structure, which is competent for recruiting chromatin-modifying enzymes. These enzymes would mediate the well-characterized activity of the LCR to modulate chromatin structure.
The Atomic Bomb Casualty Commission in retrospect
Authors: Putnam Frank W.
Published: July 2002

For 50 years, the Atomic Bomb Casualty Commission (ABCC) and its
successor, the Radiation Effects Research Foundation (RERF), have
conducted epidemiological and genetic studies of the survivors of the
atomic bombs and of their children. This research program has provided
the primary basis for radiation health standards. Both ABCC
(1947–1975) and RERF (1975 to date) have been a joint enterprise of
the United States (through the National Academy of Sciences) and of
Japan. ABCC began in devastated, occupied Japan. Its mission had to be
defined and refined. Early research revealed the urgent need for long
term study. In 1946, a Directive of President Truman enjoined the
National Research Council of the National Academy of Sciences to
develop the program. By 1950, ABCC staff exceeded 1,000, and clinical
and genetic studies were underway. Budgetary difficulties and other
problems almost forced closure in 1953. In 1955, the Francis Report led
to a unified epidemiological study. Much progress was made in the next
decade, but changing times required founding of a binational nonprofit
organization (RERF) with equal participation by Japan and the United
States. New programs have been developed and existing ones have been
extended in what is the longest continuing health survey ever
undertaken.
PU.1 as an essential activator for the expression of gp91phoxgene in human peripheral neutrophils, monocytes, and B lymphocytes
Authors: Suzuki Shoichi , Kumatori Atsushi , Haagen Inez-Anne , Fujii Yoshito , Sadat Mohamed Anowar , Jun Hao Li , Tsuji Yoshiro , Roos Dirk , Nakamura Michio
Published: July 2002

We have reported a deficiency of a 91-kDa glycoprotein component of the phagocyte NADPH oxidase (gp91phox) in neutrophils, monocytes, and B lymphocytes of a patient with X chromosome-linked chronic granulomatous disease. Sequence analysis of his gp91phoxgene revealed a single-base mutation (C → T) at position −53. Electrophoresis mobility-shift assays showed that both PU.1 and hematopoietic-associated factor 1 (HAF-1) bound to the inverted PU.1 consensus sequence centered at position −53 of the gp91phoxpromoter, and the mutation at position −53 strongly inhibited the binding of both factors. It was also indicated that a mutation at position −50 strongly inhibited PU.1 binding but hardly inhibited HAF-1 binding, and a mutation at position −56 had an opposite binding specificity for these factors. In transient expression assay using HEL cells, which express PU.1 and HAF-1, the mutations at positions −53 and −50 significantly reduced the gp91phoxpromoter activity; however, the mutation at position −56 did not affect the promoter activity. In transient cotransfection study, PU.1 dramatically activated the gp91phoxpromoter in Jurkat T cells, which originally contained HAF-1 but not PU.1. In addition, the single-base mutation (C → T) at position −52 that was identified in a patient with chronic granulomatous disease inhibited the binding of PU.1 to the promoter. We therefore conclude that PU.1 is an essential activator for the expression of gp91phoxgene in human neutrophils, monocytes, and B lymphocytes.
The interaction of Arp2/3 complex with actin: Nucleation, high affinity pointed end capping, and formation of branching networks of filaments
Authors: Mullins R. Dyche , Heuser John A. , Pollard Thomas D.
Published: July 2002

The Arp2/3 complex is a stable assembly of seven protein subunits including two actin-related proteins (Arp2 and Arp3) and five novel proteins. Previous work showed that this complex binds to the sides of actin filaments and is concentrated at the leading edges of motile cells. Here, we show that Arp2/3 complex purified from
Acanthamoeba
caps the pointed ends of actin filaments with high affinity. Arp2/3 complex inhibits both monomer addition and dissociation at the pointed ends of actin filaments with apparent nanomolar affinity and increases the critical concentration for polymerization at the pointed end from 0.6 to 1.0 μM. The high affinity of Arp2/3 complex for pointed ends and its abundance in amoebae suggest that
in vivo
all actin filament pointed ends are capped by Arp2/3 complex. Arp2/3 complex also nucleates formation of actin filaments that elongate only from their barbed ends. From kinetic analysis, the nucleation mechanism appears to involve stabilization of polymerization intermediates (probably actin dimers). In electron micrographs of quick-frozen, deep-etched samples, we see Arp2/3 bound to sides and pointed ends of actin filaments and examples of Arp2/3 complex attaching pointed ends of filaments to sides of other filaments. In these cases, the angle of attachment is a remarkably constant 70 ± 7°. From these
in vitro
biochemical properties, we propose a model for how Arp2/3 complex controls the assembly of a branching network of actin filaments at the leading edge of motile cells.
Superoxide generation by endothelial nitric oxide synthase: The influence of cofactors
Authors: Vásquez-Vivar Jeannette , Kalyanaraman B. , Martásek Pavel , Hogg Neil , Masters Bettie Sue Siler , Karoui Hakim , Tordo Paul , Pritchard Kirkwood A.
Published: July 2002

The mechanism of superoxide generation by endothelial nitric oxide synthase (eNOS) was investigated by the electron spin resonance spin-trapping technique using 5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide. In the absence of calcium/calmodulin, eNOS produces low amounts of superoxide. Upon activating eNOS electron transfer reactions by calcium/calmodulin binding, superoxide formation is increased. Heme-iron ligands, cyanide, imidazole, and the phenyl(diazene)-derived radical inhibit superoxide generation. No inhibition is observed after addition of
l
-arginine,
N
G
-hydroxy-
l
-arginine,
l
-thiocitrulline, and
l
-
N
G
-monomethyl arginine to activated eNOS. These results demonstrate that superoxide is generated from the oxygenase domain by dissociation of the ferrous–dioxygen complex and that occupation of the
l
-arginine binding site does not inhibit this process. However, the concomitant addition of
l
-arginine and tetrahydrobiopterin (BH
4
) abolishes superoxide generation by eNOS. Under these conditions,
l
-citrulline production is close to maximal. Our data indicate that BH
4
fully couples
l
-arginine oxidation to NADPH consumption and prevents dissociation of the ferrous–dioxygen complex. Under these conditions, eNOS does not generate superoxide. The presence of flavins, at concentrations commonly employed in NOS assay systems, enhances superoxide generation from the reductase domain. Our data indicate that modulation of BH
4
concentration may regulate the ratio of superoxide to nitric oxide generated by eNOS.
Antigenicity of fullerenes: Antibodies specific for fullerenes and their characteristics
Authors: Chen B.-X. , Wilson S. R. , Das M. , Coughlin D. J. , Erlanger B. F.
Published: July 2002

The recent interest in using Buckminsterfullerene (fullerene) derivatives in biological systems raises the possibility of their assay by immunological procedures. This, in turn, leads to the question of the ability of these unprecedented polygonal structures, made up solely of carbon atoms, to induce the production of specific antibodies. Immunization of mice with a C60fullerene derivative conjugated to bovine thyroglobulin yielded a population of fullerene-specific antibodies of the IgG isotype, showing that the immune repertoire was diverse enough to recognize and process fullerenes as protein conjugates. The population of antibodies included a subpopulation that crossreacted with a C70fullerene as determined by immune precipitation and ELISA procedures. These assays were made possible by the synthesis of water-soluble fullerene derivatives, including bovine and rabbit serum albumin conjugates and derivatives of trilysine and pentalysine, all of which were characterized as to the extent of substitution and their UV-Vis spectra. Possible interactions of fullerenes with the combining sites of IgG are discussed based on the physical chemistry of fullerenes and previously described protein-fullerene interactions. They remain to be confirmed by the isolation of mAbs for x-ray crystallographic studies.
Loss of social behaviors byMyxococcus xanthusduring evolution in an unstructured habitat
Authors: Velicer Gregory J. , Kroos Lee , Lenski Richard E.
Published: July 2002

Social behaviors are often targets of natural selection among higher organisms, but quantifying the effects of such selection is difficult. We have used the bacteriumMyxococcus xanthusas a model system for studying the evolution of social interactions. Changes in the social behaviors of 12M. xanthuspopulations were quantified after 1,000 generations of evolution in a liquid habitat, in which interactions among individuals were continually hindered by shaking and low cell densities. Derived lineages were compared with their ancestors with respect to maximum growth rate, motility rates on hard and soft agar, fruiting body formation ability, and sporulation frequency during starvation. Improved performance in the liquid selective regime among evolved lines was usually associated with significant reductions in all of the major social behaviors ofM. xanthus.Maintenance of functional social behaviors is apparently detrimental to fitness under asocial growth conditions.
Major histocompatibility complex class I-restricted T cells are required for all but the end stages of diabetes development in nonobese diabetic mice and use a prevalent T cell receptor α chain gene rearrangement
Authors: DiLorenzo Teresa P. , Graser Robert T. , Ono Toshiro , Christianson Gregory J. , Chapman Harold D. , Roopenian Derry C. , Nathenson Stanley G. , Serreze David V.
Published: July 2002

Nonobese diabetic (NOD) mice develop insulin-dependent diabetes mellitus due to autoimmune T lymphocyte-mediated destruction of pancreatic β cells. Although both major histocompatibility complex class I-restricted CD8+and class II-restricted CD4+T cell subsets are required, the specific role each subset plays in the pathogenic process is still unclear. Here we show that class I-dependent T cells are required for all but the terminal stages of autoimmune diabetes development. To characterize the diabetogenic CD8+T cells responsible, we isolated and propagatedin vitroCD8+T cells from the earliest insulitic lesions of NOD mice. They were cytotoxic to NOD islet cells, restricted to H-2Kd, and showed a diverse T cell receptor β chain repertoire. In contrast, their α chain repertoire was more restricted, with a recurrent amino acid sequence motif in the complementarity-determining region 3 loop and a prevalence of Vα17 family members frequently joined to the Jα42 gene segment. These results suggest that a number of the CD8+T cells participating in the initial phase of autoimmune β cell destruction recognize a common structural component of Kd/peptide complexes on pancreatic β cells, possibly a single peptide.
Pathogenic implications of mutations in the tau gene in pallido-ponto-nigral degeneration and related neurodegenerative disorders linked to chromosome 17
Authors: Clark Lorraine N. , Poorkaj Parvoneh , Wszolek Zbigniew , Geschwind Daniel H. , Nasreddine Ziad S. , Miller Bruce , Li Diane , Payami Haydeh , Awert Fre , Markopoulou Katerina , Andreadis Athena , D’Souza Ian , Lee Virginia M.-Y. , Reed Lee , Trojanowski John Q. , Zhukareva Victoria , Bird Thomas , Schellenberg Gerard , Wilhelmsen Kirk C.
Published: July 2002

Pallido-ponto-nigral degeneration (PPND) is one of the most well characterized familial neurodegenerative disorders linked to chromosome 17q21–22. These hereditary disorders are known collectively as frontotemporal dementia (FTD) and parkinsonism linked to chromosome 17 (FTDP-17). Although the clinical features and associated regional variations in the neuronal loss observed in different FTDP-17 kindreds are diverse, the diagnostic lesions of FTDP-17 brains are tau-rich filaments in the cytoplasm of specific subpopulations of neurons and glial cells. The microtubule associated protein (tau) gene is located on chromosome 17q21–22. For these reasons, we investigated the possibility that PPND and other FTDP-17 syndromes might be caused by mutations in the tau gene. Two missense mutations in exon 10 of the tau gene that segregate with disease,
Asn
279
Lys
in the PPND kindred and
Pro
301
Leu
in four other FTDP-17 kindreds, were found. A third mutation was found in the intron adjacent to the 3′ splice site of exon 10 in patients from another FTDP-17 family. Transcripts that contain exon 10 encode tau isoforms with four microtubule (MT)-binding repeats (4Rtau) as opposed to tau isoforms with three MT-binding repeats (3Rtau). The insoluble tau aggregates isolated from brains of patients with each mutation were analyzed by immunoblotting using tau-specific antibodies. For each of three mutations, abnormal tau with an apparent M
r
of 64 and 69 was observed. The dephosphorylated material comigrated with tau isoforms containing exon 10 having four MT-binding repeats but not with 3Rtau. Thus, the brains of patients with both the missense mutations and the splice junction mutation contain aggregates of insoluble 4Rtau in filamentous inclusions, which may lead to neurodegeneration.
Functional redundancy of acetylcholinesterase and neuroligin in mammalian neuritogenesis
Authors: Grifman Mirta , Galyam Nilly , Seidman Shlomo , Soreq Hermona
Published: July 2002

Accumulated evidence attributes noncatalytic morphogenic activitie(s) to acetylcholinesterase (AChE). Despite sequence homologies, functional overlaps between AChE and catalytically inactive AChE-like cell surface adhesion proteins have been demonstrated only for theDrosophilaprotein neurotactin. Furthermore, no mechanism had been proposed to enable signal transduction by AChE, an extracellular enzyme. Here, we report impaired neurite outgrowth and loss of neurexin Iα mRNA under antisense suppression of AChE in PC12 cells (AS-ACHE cells). Neurite growth was partially rescued by addition of recombinant AChE to the solid substrate or by transfection with various catalytically active and inactive AChE variants. Moreover, overexpression of the homologous neurexin I ligand, neuroligin-1, restored both neurite extension and expression of neurexin Iα. Differential PCR display revealed expression of a novel gene, nitzin, in AS-ACHE cells. Nitzin displays 42% homology to the band 4.1 protein superfamily capable of linking integral membrane proteins to the cytoskeleton. Nitzin mRNA is high throughout the developing nervous system, is partially colocalized with AChE, and increases in rescued AS-ACHE cells. Our findings demonstrate redundant neurite growth-promoting activities for AChE and neuroligin and implicate interactions of AChE-like proteins and neurexins as potential mediators of cytoarchitectural changes supporting neuritogenesis.