bluuebunny/crossref_metadata_2025 huggingface dataset:
Cloning and characterization of a histone deacetylase, HDAC9
Authors: Zhou Xianbo , Marks Paul A. , Rifkind Richard A. , Richon Victoria M.
Published: July 2002

Histone deacetylase (HDAC) catalyzes the removal of the acetyl group from the lysine residues in the N-terminal tails of nucleosomal core histones. Eight human HDACs have been identified so far. Here, we report the identification of a ninth member of the HDAC family, designated HDAC9. HDAC9 is a class II HDAC and its gene resides on human chromosome 7. HDAC9 has several alternatively spliced isoforms. One of these isoforms is histone deacetylase-related protein or myocyte enhancer-binding factor 2-interacting transcriptional repressor that we and others have previously reported and which does not possess an HDAC catalytic domain. The longest of the HDAC9 isoforms contains 1,011 aa. The isoform, designated HDAC9a, is 132 aa shorter at the C terminus than HDAC9. Also, we have identified isoforms of HDAC9 that lack the nuclear localization signal. Similar to histone deacetylase-related protein, HDAC9 transcripts are expressed at high levels in brain and skeletal muscle. The ratio of HDAC9 and HDAC9a transcripts differs among the tissues examined. HDAC9 and HDAC9a contain the HDAC catalytic domain, and Flag-tagged HDAC9 and HDAC9a possess deacetylase activity. HDAC9 and HDAC9a also repress myocyte enhancer-binding factor 2-mediated transcription. In the present study, we have identified HDAC9 and a number of alternatively spliced isoforms of HDAC9 with potentially different biological activities.
A computational analysis of sequence features involved in recognition of short introns
Authors: Lim Lee P. , Burge Christopher B.
Published: July 2002

Splicing of short introns by the nuclear pre-mRNA splicing
machinery is thought to proceed via an “intron definition”
mechanism, in which the 5′ and 3′ splice sites (5′ss, 3′ss,
respectively) are initially recognized and paired across the intron.
Here, we describe a computational analysis of sequence features
involved in recognition of short introns by using available transcript
data from five eukaryotes with complete or nearly complete genomic
sequences. The information content of five different transcript
features was measured by using methods from information theory, and
Monte Carlo simulations were used to determine the amount of
information required for accurate recognition of short introns in each
organism. We conclude: (
i
) that short introns in
Drosophila melanogaster
and
Caenorhabditis elegans
contain
essentially all of the information for their recognition by the
splicing machinery, and computer programs that simulate splicing
specificity can predict the exact boundaries of ≈95% of short
introns in both organisms; (
ii
) that in yeast, the 5′ss,
branch signal, and 3′ss can accurately identify intron locations but do
not precisely determine the location of 3′ cleavage in every intron;
and (
iii
) that the 5′ss, branch signal, and 3′ss are not
sufficient to accurately identify short introns in plant and human
transcripts, but that specific subsets of candidate intronic enhancer
motifs can be identified in both human and
Arabidopsis
that contribute dramatically to the accuracy of splicing simulators.
“What” and “where” in the human auditory system
Authors: Alain Claude , Arnott Stephen R. , Hevenor Stephanie , Graham Simon , Grady Cheryl L.
Published: July 2002

The extent to which sound identification and sound localization depend on specialized auditory pathways was examined by using functional magnetic resonance imaging and event-related brain potentials. Participants performed an S1–S2 match-to-sample task in which S1 differed from S2 in its pitch and/or location. In the pitch task, participants indicated whether S2 was lower, identical, or higher in pitch than S1. In the location task, participants were asked to localize S2 relative to S1 (i.e., leftward, same, or rightward). Relative to location, pitch processing generated greater activation in auditory cortex and the inferior frontal gyrus. Conversely, identifying the location of S2 relative to S1 generated greater activation in posterior temporal cortex, parietal cortex, and the superior frontal sulcus. Differential task-related effects on event-related brain potentials (ERPs) were seen in anterior and posterior brain regions beginning at 300 ms poststimulus and lasting for several hundred milliseconds. The converging evidence from two independent measurements of dissociable brain activity during identification and localization of identical stimuli provides strong support for specialized auditory streams in the human brain. These findings are analogous to the “what” and “where” segregation of visual information processing, and suggest that a similar functional organization exists for processing information from the auditory modality.
Y chromosomal fertility factors
kl-2
and
kl-3
of
Drosophila melanogaster
encode dynein heavy chain polypeptides
Authors: Carvalho Antonio Bernardo , Lazzaro Brian P. , Clark Andrew G.
Published: July 2002

The molecular identity and function of the
Drosophila
melanogaster
Y-linked fertility factors have long eluded
researchers. Although the
D. melanogaster
genome
sequence was recently completed, the fertility factors still were not
identified, in part because of low cloning efficiency of
heterochromatic Y sequences. Here we report a method for iterative
blast
searching to assemble heterochromatic genes from
shotgun assemblies, and we successfully identify
kl-2
and
kl-3
as 1β- and γ-dynein heavy chains,
respectively. Our conclusions are supported by formal genetics with X-Y
translocation lines. Reverse transcription–PCR was successful in
linking together unmapped sequence fragments from the whole-genome
shotgun assembly, although some sequences were missing altogether from
the shotgun effort and had to be generated
de novo
. We
also found a previously undescribed Y gene, polycystine-related
(
PRY
). The closest paralogs of
kl-2
,
kl-3
, and
PRY
(and also of
kl-5
) are autosomal and not X-linked, suggesting that
the evolution of the
Drosophila
Y chromosome has been
driven by an accumulation of male-related genes arising
de
novo
from the autosomes.
Evolutionary relationships among self-incompatibility RNases
Authors: Igic Boris , Kohn Joshua R.
Published: July 2002

T2-type RNases are responsible for self-pollen recognition and rejection in three distantly related families of flowering plants—the Solanaceae, Scrophulariaceae, and Rosaceae. We used phylogenetic analyses of 67 T2-type RNases together with information on intron number and position to determine whether the use of RNases for self-incompatibility in these families is homologous or convergent. All methods of phylogenetic reconstruction as well as patterns of variation in intron structure find that all self-incompatibility RNases along with non-S genes from only two taxa form a monophyletic clade. Several lines of evidence suggest that the best interpretation of this pattern is homology of self-incompatibility RNases from the Scrophulariaceae, Solanaceae, and Rosaceae. Because the most recent common ancestor of these three families is the ancestor of ≈75% of dicot families, our results indicate that RNase-based self-incompatibility was the ancestral state in the majority of dicots.
Radiation risk to low fluences of α particles may be greater than we thought
Authors: Zhou Hongning , Suzuki Masao , Randers-Pehrson Gerhard , Vannais Diane , Chen Gang , Trosko James E. , Waldren Charles A. , Hei Tom K.
Published: July 2002

Based principally on the cancer incidence found in survivors
of the atomic bombs dropped in Hiroshima and Nagasaki, the
International Commission on Radiation Protection (ICRP) and the United
States National Council on Radiation Protection and Measurements (NCRP)
have recommended that estimates of cancer risk for low dose exposure be
extrapolated from higher doses by using a linear, no-threshold model.
This recommendation is based on the dogma that the DNA of the nucleus
is the main target for radiation-induced genotoxicity and, as fewer
cells are directly damaged, the deleterious effects of radiation
proportionally decline. In this paper, we used a precision microbeam to
target an exact fraction (either 100% or ≤20%) of the cells in a
confluent population and irradiated their nuclei with exactly one α
particle each. We found that the frequencies of induced mutations and
chromosomal changes in populations where some known fractions of nuclei
were hit are consistent with non-hit cells contributing significantly
to the response. In fact, irradiation of 10% of a confluent mammalian
cell population with a single α particle per cell results in a mutant
yield similar to that observed when all of the cells in the population
are irradiated. This effect was significantly eliminated in cells
pretreated with a 1 mM dose of octanol, which inhibits gap
junction-mediated intercellular communication, or in cells carrying a
dominant negative connexin 43 vector. The data imply that the relevant
target for radiation mutagenesis is larger than an individual cell and
suggest a need to reconsider the validity of the linear extrapolation
in making risk estimates for low dose, high
linear-energy-transfer (LET) radiation exposure.
Gene expression in papillary thyroid carcinoma reveals highly consistent profiles
Authors: Huang Ying , Prasad Manju , Lemon William J. , Hampel Heather , Wright Fred A. , Kornacker Karl , LiVolsi Virginia , Frankel Wendy , Kloos Richard T. , Eng Charis , Pellegata Natalia S. , de la Chapelle Albert
Published: July 2002

Papillary thyroid carcinoma (PTC) is clinically
heterogeneous. Apart from an association with ionizing
radiation, the etiology and molecular biology of PTC is poorly
understood. We used oligo-based DNA arrays to study the expression
profiles of eight matched pairs of normal thyroid and PTC tissues.
Additional PTC tumors and other tissues were studied by reverse
transcriptase–PCR and immunohistochemistry. The PTCs showed concordant
expression of many genes and distinct clustered profiles. Genes with
increased expression in PTC included many encoding adhesion and
extracellular matrix proteins. Expression was increased in 8/8 tumors
for 24 genes and in 7/8 tumors for 22 genes. Among these genes were
several previously known to be overexpressed in PTC, such as
MET
,
LGALS3
,
KRT19
,
DPP4
,
MDK
,
TIMP1
, and
FN1
. The numerous additional genes include
CITED1
,
CHI3L1
,
ODZ1
,
N33
,
SFTPB
, and
SCEL
.
Reverse transcriptase–PCR showed high expression of
CITED1
,
CHI3L1
,
ODZ1
, and
SCEL
in 6/6 additional PTCs. Immunohistochemical
analysis detected CITED1 and SFTPB in 49/52 and 39/52 PTCs,
respectively, but not in follicular thyroid carcinoma and normal
thyroid tissue. Genes underexpressed in PTC included tumor suppressors,
thyroid function-related proteins, and fatty acid binding proteins.
Expression was decreased in 7/8 tumors for eight genes and decreased
in 6/8 tumors for 19 genes. We conclude that, despite its clinical
heterogeneity, PTC is characterized by consistent and specific
molecular changes. These findings reveal clues to the molecular
pathways involved in PTC and may provide biomarkers for clinical use.
Interaction of cyclooxygenases with an apoptosis- and autoimmunity-associated protein.
Authors: Ballif B A , Mincek N V , Barratt J T , Wilson M L , Simmons D L
Published: July 2002

Cyclooxygenases (COXs) 1 and 2 are 72-kDa, intralumenal residents of the endoplasmic reticulum (ER) and nuclear envelope, where they catalyze the rate-limiting steps in the conversion of arachidonate to the physiologically dynamic prostanoids. Recent studies, including the generation of knockout mice, show COX-1 and COX-2 to have biologically distinct roles within cells and organisms. Also apparent is that arachidonate substrate is selectably metabolized by COX-2 after mitogen stimulation in many cells that contain both isoforms. Because COX-1 and COX-2 are highly conserved in all residues needed for catalysis and in their purified forms have almost identical kinetic properties, we have searched for COX-interacting ER proteins that might mediate these unique isoenzymic properties. Using COXs as bait in the yeast two-hybrid system, we identified autoimmunity- and apoptosis-associated nucleobindin (Nuc) as a protein that specifically interacts with both isoenzymes. COX-Nuc binding was substantiated by immunoprecipitation experiments, which showed that COX-1 and, to a lesser extent, COX-2 form complexes with Nuc in vitro. When overexpressed in COS-1 cells, Nuc was found to be extracellularly released. However, when Nuc was co-overexpressed with COX-1 or COX-2, its release was reduced by >80%. This finding suggests that COX isoenzymes participate in the retention of Nuc within the lumen of the ER, where COX may regulate the release of Nuc from the cell. It also identifies Nuc as a potential regulator of COXs through this interaction.
The glycine binding site of the N-methyl-D-aspartate receptor subunit NR1: identification of novel determinants of co-agonist potentiation in the extracellular M3-M4 loop region.
Authors: Hirai H , Kirsch J , Laube B , Betz H , Kuhse J
Published: July 2002

The N-methyl-D-aspartate (NMDA) subtype of ionotropic glutamate receptors is a heterooligomeric membrane protein composed of homologous subunits. Here, the contribution of the M3-M4 loop of the NR1 subunit to the binding of glutamate and the co-agonist glycine was investigated by site-directed mutagenesis. Substitution of the phenylalanine residues at positions 735 or 736 of the M3-M4 loop produced a 15- to 30-fold reduction in apparent glycine affinity without affecting the binding of glutamate and the competitive glycine antagonist 7-chlorokynurenic acid; mutation of both residues caused a >100-fold decrease in glycine affinity. These residues are found in a C-terminal region of the M3-M4 loop that shows significant sequence similarity to bacterial amino acid-binding proteins. Epitope tagging revealed both the N-terminus and the M3-M4 loop to be exposed extracellularly, whereas a C-terminal epitope was localized intracellularly. These results indicate that the M3-M4 loop is part of the ligand-binding pocket of the NR1 subunit and provide the basis for a refined model of the glycine-binding site of the NMDA receptor.
Neuronal nitric oxide synthase and dystrophin-deficient muscular dystrophy.
Authors: Chang W J , Iannaccone S T , Lau K S , Masters B S , McCabe T J , McMillan K , Padre R C , Spencer M J , Tidball J G , Stull J T
Published: July 2002

Neuronal nitric oxide synthase (nNOS) in fast-twitch skeletal muscle fibers is primarily particulate in contrast to its greater solubility in brain. Immunohistochemistry shows nNOS localized to the sarcolemma, with enrichment at force transmitting sites, the myotendinous junctions, and costameres. Because this distribution is similar to dystrophin, we determined if nNOS expression was affected by the loss of dystrophin. Significant nNOS immunoreactivity and enzyme activity was absent in skeletal muscle tissues from patients with Duchenne muscular dystrophy. Similarly, in dystrophin-deficient skeletal muscles from mdx mice both soluble and particulate nNOS was greatly reduced compared with C57 control mice. nNOS mRNA was also reduced in mdx muscle in contrast to mRNA levels for a dystrophin binding protein, alpha 1-syntrophin. nNOS levels increased dramatically from 2 to 52 weeks of age in C57 skeletal muscle, which may indicate a physiological role for NO in aging-related processes. Biochemical purification readily dissociates nNOS from the dystrophin-glycoprotein complex. Thus, nNOS is not an integral component of the dystrophin-glycoprotein complex and is not simply another dystrophin-associated protein since the expression of both nNOS mRNA and protein is affected by dystrophin expression.
Chronic control of high blood pressure in the spontaneously hypertensive rat by delivery of angiotensin type 1 receptor antisense.
Authors: Iyer S N , Lu D , Katovich M J , Raizada M K
Published: July 2002

The renin-angiotensin system plays a crucial role in the development and establishment of the hypertensive state in the spontaneously hypertensive (SH) rat. Interruption of this system's activity by pharmacological means results in the lowering of blood pressure (BP) and control of hypertension. However, such means are temporary and require the continuous use of drugs for the control of this pathophysiological state. Our objective in this investigation was to determine if a virally mediated gene-transfer approach using angiotensin type 1 receptor antisense (AT1R-AS) could be used to control hypertension on a long-term basis in the SH rat model of human essential hypertension. Injection of viral particles containing AT1R-AS (LNSV-AT1R-AS) in 5-day-old rats resulted in a lowering of BP exclusively in the SH rat and not in the Wistar Kyoto normotensive control. A maximal anti-hypertensive response of 33 +/- 5 mmHg was observed, was maintained throughout development, and still persisted 3 months after administration of LNSV-AT1R-AS. The lowering of BP was associated with the expression of AT1R-AS transcript and decreases in AT1-receptor in many peripheral angiotensin II target tissues such as mesenteric artery, adrenal gland, heart, and kidney. Attenuation of angiotensin II-stimulated physiological actions such as contraction of aortic rings and increase in BP was also observed in the LNSV-AT1R-AS-treated SH rat. These observations show that a single injection of LNSV-AT1R-AS normalizes BP in the SH rat on a long-term basis. They suggest that such a gene-transfer strategy can be successfully used to control the development of hypertension on a permanent basis.
Activation domain-mediated enhancement of activator binding to chromatin in mammalian cells.
Authors: Bunker C A , Kingston R E
Published: July 2002

DNA binding by transcriptional activators is typically an obligatory step in the activation of gene expression. Activator binding and subsequent steps in transcription are repressed by genomic chromatin. Studies in vitro have suggested that overcoming this repression is an important function of some activation domains. Here we provide quantitative in vivo evidence that the activation domain of GAL4-VP16 can increase the affinity of GAL4 for its binding site on genomic DNA in mammalian cells. Moreover, the VP16 activation domain has a much greater stimulatory effect on expression from a genomic reporter gene than on a transiently transfected reporter gene, where factor binding is more permissive. We found that not all activation domains showed a greater activation potential in a genomic context, suggesting that only some activation domains can function in vivo to alleviate the repressive effects of chromatin. These data demonstrate the importance of activation domains in relieving chromatin-mediated repression in vivo and suggest that one way they function is to increase binding of the activator itself.
Mechanism of oligonucleotide release from cationic liposomes.
Authors: Zelphati O , Szoka F C
Published: July 2002

We propose a mechanism for oligonucleotide (ODN) release from cationic lipid complexes in cells that accounts for various observations on cationic lipid-nucleic acid-cell interactions. Fluorescent confocal microscopy of cells treated with rhodamine-labeled cationic liposome/ fluorescein-labeled ODN (F-ODN) complexes show the F-ODN separates from the lipid after internalization and enters the nucleus leaving the fluorescent lipid in cytoplasmic structures. ODN displacement from the complex was studied by fluorescent resonance energy transfer. Anionic liposome compositions (e.g., phosphatidylserine) that mimic the cytoplasmic facing monolayer of the cell membrane released ODN from the complex at about a 1:1 (-/+) charge ratio. Release was independent of ionic strength and pH. Physical separation of the F-ODN from monovalent and multivalent cationic lipids was confirmed by gel electrophoresis. Fluid but not solid phase anionic liposomes are required, whereas the physical state of the cationic lipids does not effect the release. Water soluble molecules with a high negative linear charge density, dextran sulfate, or heparin also release ODN. However, ATP, spermidine, spermine, tRNA, DNA, polyglutamic acid, polylysine, bovine serum albumin, or histone did not release ODN, even at 100-fold charge excess (-/+). Based upon these results, we propose that the complex, after internalization by endocytosis, induces flip-flop of anionic lipids from the cytoplasmic facing monolayer. Anionic lipids laterally diffuse into the complex and form a charged neutralized ion-pair with the cationic lipids. This leads to displacement of the ODN from the cationic lipid and its release into the cytoplasm.
Phosphorylation of synaptic vesicle proteins: modulation of the alpha SNAP interaction with the core complex.
Authors: Hirling H , Scheller R H
Published: July 2002

We analyzed whether synaptic membrane trafficking proteins are substrates for casein kinase II, calcium/calmodulin-dependent protein kinase II, and cAMP-dependent protein kinase (PKA), three kinases implicated in the modulation of synaptic transmission. Each kinase phosphorylates a specific set of the vesicle proteins syntaxin 1A, N-ethylmaleimide-sensitive factor (NSF), vesicle-associated membrane protein (VAMP), synaptosome-associated 25-kDa protein (SNAP-25), n-sec1, alpha soluble NSF attachment protein (alpha SNAP), and synaptotagmin. VAMP is phosphorylated by calcium/calmodulin-dependent protein kinase II on serine 61. alpha SNAP is phosphorylated by PKA; however, the beta SNAP isoform is phosphorylated only 20% as efficiently. alpha SNAP phosphorylated by PKA binds to the core docking and fusion complex 10 times weaker than the dephosphorylated form. These studies provide a first glimpse at regulatory events that may be important in modulating neurotransmitter release during learning and memory.
A model of multiplicative neural responses in parietal cortex.
Authors: Salinas E , Abbott L F
Published: July 2002

Visual responses of neurons in parietal area 7a are modulated by a combined eye and head position signal in a multiplicative manner. Neurons with multiplicative responses can act as powerful computational elements in neural networks. In the case of parietal cortex, multiplicative gain modulation appears to play a crucial role in the transformation of object locations from retinal to body-centered coordinates. It has proven difficult to uncover single-neuron mechanisms that account for neuronal multiplication. Here we show that multiplicative responses can arise in a network model through population effects. Specifically, neurons in a recurrently connected network with excitatory connections between similarly tuned neurons and inhibitory connections between differently tuned neurons can perform a product operation on additive synaptic inputs. The results suggest that parietal responses may be based on this architecture.
Cell stress-regulated human major histocompatibility complex class I gene expressed in gastrointestinal epithelium.
Authors: Groh V , Bahram S , Bauer S , Herman A , Beauchamp M , Spies T
Published: July 2002

Conventional major histocompatibility complex (MHC) class I genes encode molecules that present intracellular peptide antigens to T cells. They are ubiquitously expressed and regulated by interferon gamma. Two highly divergent human MHC class I genes, MICA and MICB, are regulated by promoter heat shock elements similar to those of HSP70 genes. MICA encodes a cell surface glycoprotein, which is not associated with beta 2-microglobulin, is conformationally stable independent of conventional class I peptide ligands, and almost exclusively expressed in gastrointestinal epithelium. Thus, this MHC class I molecule may function as an indicator of cell stress and may be recognized by a subset of gut mucosal T cells in an unusual interaction.
Disappearance of body fat in normal rats induced by adenovirus-mediated leptin gene therapy
Authors: Chen Guoxun , Koyama Kazunori , Yuan Xue , Lee Young , Zhou Yan-Ting , O’Doherty Robert , Newgard Christopher B. , Unger Roger H.
Published: July 2002

Sustained hyperleptinemia of 8 ng/ml was induced for 28
days in normal Wistar rats by infusing a recombinant adenovirus
containing the rat leptin cDNA (AdCMV-leptin). Hyperleptinemic rats
exhibited a 30–50% reduction in food intake and gained only 22 g
over the experimental period versus 115–132 g in control animals that
received saline infusions or a recombinant virus containing the
β-galactosidase gene (AdCMV-βGal). Body fat was absent in
hyperleptinemic rats, whereas control rats pair-fed to the
hyperleptinemic rats retained ≈50% body fat. Further, plasma
triglycerides and insulin levels were significantly lower in
hyperleptinemic versus pair-fed controls, while fatty acid and glucose
levels were similar in the two groups, suggestive of enhanced insulin
sensitivity in the hyperleptinemic animals. Thus, despite equivalent
reductions in food intake and weight gain in hyperleptinemic and
pair-fed animals, identifiable fat tissue was completely ablated only
in the former group, raising the possibility of a specific lipoatrophic
activity for leptin.
The gcm-motif: A novel DNA-binding motif conserved in
Drosophila
and mammals
Authors: Akiyama Yasuko , Hosoya Toshihiko , Poole Anthony M. , Hotta Yoshiki
Published: July 2002

In the
Drosophila
nervous system, the
glial
cells missing
gene (
gcm
) is transiently
expressed in glial precursors to switch their fate from the neuronal
default to glia. It encodes a novel 504-amino acid protein with a
nuclear localization signal. We report here that the GCM protein is a
novel DNA-binding protein and that its DNA-binding activity is
localized in the N-terminal 181 amino acids. It binds with high
specificity to the nucleotide sequence, (A/G)CCCGCAT, which is a
novel sequence among known targets of DNA-binding proteins. Eleven such
GCM-binding sequences are found in the 5′ upstream region of the
repo
gene, whose expression in early glial cells is
dependent on
gcm
. This suggests that the GCM protein is
a transcriptional regulator directly controlling
repo
.
We have also identified homologous genes from human and mouse whose
products share a highly conserved N-terminal region with
Drosophila
GCM. At least one of these was shown to have
DNA-binding activity similar to that of GCM. By comparing the deduced
amino acid sequences of these gene products, we were able to define the
“gcm motif,” an evolutionarily conserved motif with DNA-binding
activity. By PCR amplification, we obtained evidence for the existence
of additional gcm-motif genes in mouse as well as in
Drosophila
. The gcm-motif, therefore, forms a family of
novel DNA-binding proteins, and may function in various aspects of cell
fate determination.
Phylogenetic resolution within the Elephantidae using fossil DNA sequence from the American mastodon (Mammut americanum) as an outgroup.
Authors: Yang H , Golenberg E M , Shoshani J
Published: July 2002

DNA was extracted from the extinct American mastodon, the extinct woolly mammoth, and the modern Asian and African elephants to test the traditional morphologically based phylogeny within Elephantidae. Phylogenetic analyses of the aligned sequences of the mitochondrial gene cytochrome b support a monophyletic Asian elephant-woolly mammoth clade when the American mastodon is used as an outgroup. Previous molecular studies were unable to resolve the relationships of the woolly mammoth, Asian elephant, and African elephant because the sequences appear to have evolved at heterogeneous rates and inappropriate outgroups were used for analysis. The results demonstrate the usefulness of fossil molecular data from appropriate sister taxa for resolving phylogenies of highly derived or early radiating lineages.
CD14+ blood monocytes can differentiate into functionally mature CD83+ dendritic cells.
Authors: Zhou L J , Tedder T F
Published: July 2002

Dendritic cells are potent antigen-presenting cells that initiate primary immune responses. Although dendritic cells derive from bone marrow stem cells, the intermediate stages in their development remain unknown. In this study, plastic-adherent blood monocytes (CD14+, CD1a-) cultured for 7 days with granulocyte-monocyte colony-stimulating factor, interleukin 4, and tumor necrosis factor alpha were shown to differentiate into CD1a+ CD83+ dendritic cells. These cells displayed all phenotypic and morphologic characteristics of mature dendritic cells and were the most potent stimulatory cells in allogeneic mixed leukocyte reactions. The identification of specific culture conditions that generate large numbers of dendritic cells from purified monocytes uncovers an important step in dendritic cell maturation that will allow the further characterization of their role in autoimmune diseases, graft rejection, and human immunodeficiency virus infection.
RecA-like proteins are components of early meiotic nodules in lily
Authors: Anderson Lorinda K. , Offenberg Hildo H. , Verkuijlen W. M. H. C. , Heyting Christa
Published: July 2002

Early meiotic nodules (also called recombination nodules) are proteinaceous structures about 100 nm in diameter that are associated with forming synaptonemal complexes (SCs) during early prophase I of meiosis. Although their function is unknown, early nodules may be involved in searching for DNA homology before synaptic initiation. Two potential components of early nodules are Rad51 and Dmc1 proteins. These proteins are important for meiotic recombination in eukaryotes and are homologous to RecA, the major protein that catalyzes homologous pairing and DNA strand exchange in prokaryotes. In addition, Rad51 has been localized by immunofluorescence in abundant foci that may correspond to early nodules in yeast, lily, and mouse. In yeast and lily, Dmc1 and Lim15, the lily homolog of Dmc1, colocalize with Rad51. Here, using electron microscopic immunogold localization to spreads of zygotene and early pachytene SCs from lily, we confirm that RecA-like proteins are components of early nodules. The antibody used was generated to full-length tomato Rad51 protein and binds to both Rad51 and Lim15 in immunoblots of lily primary microsporocyte proteins. The labeled early nodules are heterogeneous in size and are associated with both axial elements and SCs. There are two classes of early nodules, those that are densely labeled with gold and those that are not labeled at all. This result may be due to technical limitations associated with using spread preparations or to differences in the nodules themselves. The presence of Rad51 and/or Lim15 proteins in early nodules supports the hypothesis that early nodules are involved in recombination-related events during meiosis.
Acetylcholinesterase-transgenic mice display embryonic modulations in spinal cord choline acetyltransferase and neurexin Iβ gene expression followed by late-onset neuromotor deterioration
Authors: Andres Christian , Beeri Rachel , Friedman Alon , Lev-Lehman Efrat , Henis Sivan , Timberg Rina , Shani Moshe , Soreq Hermona
Published: July 2002

To explore the possibility that overproduction of neuronal acetylcholinesterase (AChE) confers changes in both cholinergic and morphogenic intercellular interactions, we studied developmental responses to neuronal AChE overexpression in motoneurons and neuromuscular junctions of AChE-transgenic mice. Perikarya of spinal cord motoneurons were consistently enlarged from embryonic through adult stages in AChE-transgenic mice. Atypical motoneuron development was accompanied by premature enhancement in the embryonic spinal cord expression of choline acetyltransferase mRNA, encoding the acetylcholine-synthesizing enzyme choline acetyltransferase. In contrast, the mRNA encoding for neurexin-Iβ, the heterophilic ligand of the AChE-homologous neuronal cell surface protein neuroligin, was drastically lower in embryonic transgenic spinal cord than in controls. Postnatal cessation of these dual transcriptional responses was followed by late-onset deterioration in neuromotor performance that was associated with gross aberrations in neuromuscular ultrastructure and with pronounced amyotrophy. These findings demonstrate embryonic feedback mechanisms to neuronal AChE overexpression that are attributable to both cholinergic and cell–cell interaction pathways, suggesting that embryonic neurexin Iβ expression is concerted
in vivo
with AChE levels and indicating that postnatal changes in neuronal AChE-associated proteins may be involved in late-onset neuromotor pathologies.
Expression and activation of SH2/PTB-containing ShcA adaptor protein reflects the pattern of neurogenesis in the mammalian brain
Authors: Conti Luciano , De Fraja Claudio , Gulisano Massimo , Migliaccio Enrica , Govoni Stefano , Cattaneo Elena
Published: July 2002

The adult mammalian brain comprises many functionally distinct neuronal types, which are generated during development as a result of a coordinated signaling cascade that drives neuroblasts from proliferation into differentiation. We investigated whether and how ShcA adaptor proteins, which are known to function as initiators of the Ras signaling cascade in various nonneuronal systems where they have been considered to be expressed ubiquitously, are involved in the proliferative and differentiative phases of the developing brain. We found that in the forebrain expression and activation of ShcA proteins were strictly regulated during embryonic development, both temporally and spatially. The mRNAs encoded by the
ShcA
gene were expressed exclusively within an area to which active proliferation of immature neuroblasts was confined, the ventricular zone. In postnatal and adult brain,
ShcA
mRNAs and proteins were present only faintly. In the adult olfactory epithelium, in which neuronal cell renewal occurs throughout life, ShcA remained strongly expressed. These phenomena were peculiar to ShcA, since Grb2 adaptor protein remained expressed at constant level throughout development. The embryonically expressed ShcA proteins were functionally active, since p52
ShcA
became phosphorylated on tyrosine and associated with Grb2 following intraventricular injection of epidermal growth factor in the embryonic brain. Our data indicate that, through an orderly pattern of expression,
ShcA
gene products may play a role in the control of the switch between proliferation and differentiation of brain neuroblasts.
P-TEN, the tumor suppressor from human chromosome 10q23, is a dual-specificity phosphatase
Authors: Myers Michael P. , Stolarov Javor P. , Eng Charis , Li Jing , Wang Steven I. , Wigler Michael H. , Parsons Ramon , Tonks Nicholas K.
Published: July 2002

Protein tyrosine phosphatases (PTPs) have long been thought to play a role in tumor suppression due to their ability to antagonize the growth promoting protein tyrosine kinases. Recently, a candidate tumor suppressor from 10q23, termed P-TEN, was isolated, and sequence homology was demonstrated with members of the PTP family, as well as the cytoskeletal protein tensin. Here we show that recombinant P-TEN dephosphorylated protein and peptide substrates phosphorylated on serine, threonine, and tyrosine residues, indicating that P-TEN is a dual-specificity phosphatase. In addition, P-TEN exhibited a high degree of substrate specificity, showing selectivity for extremely acidic substrates
in vitro
. Furthermore, we demonstrate that mutations in P-TEN, identified from primary tumors, tumor cells lines, and a patient with Bannayan–Zonana syndrome, resulted in the ablation of phosphatase activity, demonstrating that enzymatic activity of P-TEN is necessary for its ability to function as a tumor suppressor.
hSK4, a member of a novel subfamily of calcium-activated potassium channels
Authors: Joiner William J. , Wang Lu-Yang , Tang Michael D. , Kaczmarek Leonard K.
Published: July 2002

The gene for hSK4, a novel human small conductance calcium-activated potassium channel, or SK channel, has been identified and expressed in Chinese hamster ovary cells. In physiological saline hSK4 generates a conductance of approximately 12 pS, a value in close agreement with that of other cloned SK channels. Like other members of this family, the polypeptide encoded by hSK4 contains a previously unnoted leucine zipper-like domain in its C terminus of unknown function. hSK4 appears unique, however, in its very high affinity for Ca2+(EC50of 95 nM) and its predominant expression in nonexcitable tissues of adult animals. Together with the relatively low homology of hSK4 to other SK channel polypeptides (approximately 40% identical), these data suggest that hSK4 belongs to a novel subfamily of SK channels.
Synchronization of oscillatory responses in visual cortex correlates with perception in interocular rivalry
Authors: Fries Pascal , Roelfsema Pieter R. , Engel Andreas K. , König Peter , Singer Wolf
Published: July 2002

In subjects suffering from early onset strabismus, signals conveyed by the two eyes are not perceived simultaneously but in alternation. We exploited this phenomenon of interocular suppression to investigate the neuronal correlate of binocular rivalry in primary visual cortex of awake strabismic cats. Monocularly presented stimuli that were readily perceived by the animal evoked synchronized discharges with an oscillatory patterning in the γ-frequency range. Upon dichoptic stimulation, neurons responding to the stimulus that continued to be perceived increased the synchronicity and the regularity of their oscillatory patterning while the reverse was true for neurons responding to the stimulus that was no longer perceived. These differential changes were not associated with modifications of discharge rate, suggesting that at early stages of visual processing the degree of synchronicity rather than the amplitude of responses determines which signals are perceived and control behavioral responses.
The synthesis of ATP by glycolytic enzymes in the postsynaptic density and the effect of endogenously generated nitric oxide
Authors: Wu Kuo , Aoki Chiye , Elste Alice , Rogalski-Wilk Adrienne A. , Siekevitz Philip
Published: July 2002

The major contribution of this paper is the finding of a glycolytic source of ATP in the isolated postsynaptic density (PSD). The enzymes involved in the generation of ATP are glyceraldehyde-3-phosphate dehydrogenase (G3PD) and phosphoglycerate kinase (PGK). Lactate dehydrogenase (LDH) is available for the regeneration of NAD
+
, as well as aldolase for the regeneration of glyceraldehyde-3-phosphate (G3P). The ATP was shown to be used by the PSD Ca
2+
/calmodulin-dependent protein kinase and can probably be used by two other PSD kinases, protein kinase A and protein kinase C. We confirmed by immunocytochemistry the presence of G3PD in the PSD and its binding to actin. Also present in the PSD is NO synthase, the source of NO. NO increases the binding of NAD, a G3PD cofactor, to G3PD and inhibits its activity as also found by others. The increased NAD binding resulted in an increase in G3PD binding to actin. We confirmed the autophosphorylation of G3PD by ATP, and further found that this procedure also increased the binding of G3PD to actin. ATP and NO are connected in that the formation of NO from NOS at the PSD resulted, in the presence of NAD, in a decrease of ATP formation in the PSD. In the discussion, we raise the possible roles of G3PD and of ATP in protein synthesis at the PSD, the regulation by NO, as well as the overall regulatory role of the PSD complex in synaptic transmission.
Brain activity in visual cortex predicts individual differences in reading performance
Authors: Demb Jonathan B. , Boynton Geoffrey M. , Heeger David J.
Published: July 2002

The relationship between brain activity and reading performance was examined to test the hypothesis that dyslexia involves a deficit in a specific visual pathway known as the magnocellular (M) pathway. Functional magnetic resonance imaging was used to measure brain activity in dyslexic and control subjects in conditions designed to preferentially stimulate the M pathway. Dyslexics showed reduced activity compared with controls both in the primary visual cortex and in a secondary cortical visual area (MT+) that is believed to receive a strong M pathway input. Most importantly, significant correlations were found between individual differences in reading rate and brain activity. These results support the hypothesis for an M pathway abnormality in dyslexia and imply a strong relationship between the integrity of the M pathway and reading ability.
Interactions of the chaperone Hsp104 with yeast Sup35 and mammalian PrP
Authors: Schirmer Eric C. , Lindquist Susan
Published: July 2002

[
PSI
+] is a genetic element in yeast for which a heritable change in phenotype appears to be caused by a heritable change in the conformational state of the Sup35 protein. The inheritance of [
PSI
+] and the physical state of Sup35
in vivo
depend on the protein chaperone Hsp104 (heat shock protein 104). Although these observations provide a strong genetic argument in support of the “protein-only” or “prion” hypothesis for [
PSI
+], there is, as yet, no direct evidence of an interaction between the two proteins. We report that when purified Sup35 and Hsp104 are mixed, the circular dichroism (CD) spectrum differs from that predicted by the addition of the proteins’ individual spectra, and the ATPase activity of Hsp104 is inhibited. Similar results are obtained with two other amyloidogenic substrates, mammalian PrP and β-amyloid 1-42 peptide, but not with several control proteins. With a group of peptides that span the PrP protein sequence, those that produced the largest changes in CD spectra also caused the strongest inhibition of ATPase activity in Hsp104. Our observations suggest that (
i
) previously described genetic interactions between Hsp104 and [
PSI
+] are caused by direct interaction between Hsp104 and Sup35; (
ii
) Sup35 and PrP, the determinants of the yeast and mammalian prions, respectively, share structural features that lead to a specific interaction with Hsp104; and (
iii
) these interactions couple a change in structure to the ATPase activity of Hsp104.
Chronic stress alters synaptic terminal structure in hippocampus
Authors: Magariños Ana María , Verdugo José M. García , McEwen Bruce S.
Published: July 2002

Repeated psychosocial or restraint stress causes atrophy of apical dendrites in CA3 pyramidal neurons of the hippocampus, accompanied by specific cognitive deficits in spatial learning and memory. Excitatory amino acids mediate this atrophy together with adrenal steroids and the neurotransmitter serotonin. Because the mossy fibers from dentate granule neurons provide a major excitatory input to the CA3 proximal apical dendrites, we measured ultrastructural parameters associated with the mossy fiber–CA3 synapses in control and 21-day restraint-stressed rats in an effort to find additional morphological consequences of stress that could help elucidate the underlying anatomical as well as cellular and molecular mechanisms. Although mossy fiber terminals of control rats were packed with small, clear synaptic vesicles, terminals from stressed animals showed a marked rearrangement of vesicles, with more densely packed clusters localized in the vicinity of active zones. Moreover, compared with controls, restraint stress increased the area of the mossy fiber terminal occupied by mitochondrial profiles and consequently, a larger, localized energy-generating capacity. A single stress session did not produce these changes either immediately after or the next day following the restraint session. These findings provide a morphological marker of the effects of chronic stress on the hippocampus that points to possible underlying neuroanatomical as well as cellular and molecular mechanisms for the ability of repeated stress to cause structural changes within the hippocampus.
Explanation by the double-metal-ion mechanism of catalysis for the differential metal ion effects on the cleavage rates of 5′-oxy and 5′-thio substrates by a hammerhead ribozyme
Authors: Zhou De-Min , Zhang Li-He , Taira Kazunari
Published: July 2002

In a previous examination using natural all-RNA substrates
that contained either a 5′-oxy or 5′-thio leaving group at the cleavage
site, we demonstrated that (
i
) the attack by the
2′-oxygen at C17 on the phosphorus atom is the rate-limiting step only
for the substrate that contains a 5′-thio group (R11S) and
(
ii
) the departure of the 5′ leaving group is the
rate-limiting step for the natural all-RNA substrate (R11O) in both
nonenzymatic and hammerhead ribozyme-catalyzed reactions; the energy
diagrams for these reactions were provided in our previous publication.
In this report we found that the rate of cleavage of R11O by a
hammerhead ribozyme was enhanced 14-fold when Mg
2+
ions
were replaced by Mn
2+
ions, whereas the rate of cleavage of
R11S was enhanced only 2.2-fold when Mg
2+
ions were
replaced by Mn
2+
ions. This result appears to be exactly
the opposite of that predicted from the direct coordination of the
metal ion with the leaving 5′-oxygen, because a switch in metal ion
specificity was not observed with the 5′-thio substrate. However, our
quantitative analyses based on the previously provided energy diagram
indicate that this result is in accord with the double-metal-ion
mechanism of catalysis.
Postnatal mouse subventricular zone neuronal precursors can migrate and differentiate within multiple levels of the developing neuraxis
Authors: Lim Daniel A. , Fishell Gord J. , Alvarez-Buylla Arturo
Published: July 2002

The mammalian subventricular zone (SVZ) of the lateral wall of the forebrain ventricle retains a population of proliferating neuronal precursors throughout life. Neuronal precursors born in the postnatal and adult SVZ migrate to the olfactory bulb where they differentiate into interneurons. Here we tested the potential of mouse postnatal SVZ precursors in the environment of the embryonic brain: (
i
) a ubiquitous genetic marker, (
ii
) a neuron-specific transgene, and (
iii
) a lipophilic-dye were used to follow the fate of postnatal day 5–10 SVZ cells grafted into embryonic mouse brain ventricles at day 15 of gestation. Graft-derived cells were found at multiple levels of the neuraxis, including septum, thalamus, hypothalamus, and in large numbers in the midbrain inferior colliculus. We observed no integration into the cortex. Neuronal differentiation of graft derived cells was demonstrated by double-staining with neuron-specific β-tubulin antibodies, expression of the neuron-specific transgene, and the dendritic arbors revealed by the lipophilic dye. We conclude that postnatal SVZ cells can migrate through and differentiate into neurons within multiple embryonic brain regions other than the olfactory bulb.