bluuebunny/crossref_metadata_2025 huggingface dataset:
Long-term functional recovery from age-induced spatial memory impairments by nerve growth factor gene transfer to the rat basal forebrain.
Authors: Martínez-Serrano A , Fischer W , Söderström S , Ebendal T , Björklund A
Published: July 2002

Nerve growth factor (NGF) stimulates functional recovery from cognitive impairments associated with aging, either when administered as a purified protein or by means of gene transfer to the basal forebrain. Because gene transfer procedures need to be tested in long-term experimental paradigms to assess their in vivo efficiency, we have used ex vivo experimental gene therapy to provide local delivery of NGF to the aged rat brain over a period of 2.5 months by transplanting immortalized central nervous system-derived neural stem cells genetically engineered to secrete NGF. By grafting them at two independent locations in the basal forebrain, medial septum and nucleus basalis magnocellularis, we show that functional recovery as assessed in the Morris water maze can be achieved by neurotrophic stimulation of any of these cholinergic cell groups. Moreover, the cholinergic neurons in the grafted regions showed a hypertrophic response resulting in a reversal of the age-associated atrophy seen in the learning-impaired aged control rats. Long-term expression of the transgene lead to an increased NGF tissue content (as determined by NGF-ELISA) in the transplanted regions up to at least 10 weeks after grafting. We conclude that the gene transfer procedure used here is efficient to provide the brain with a long-lasting local supply of exogenous NGF, induces long-term functional recovery of cognitive functions, and that independent trophic stimulation of the medial septum or nucleus basalis magnocellularis has similar consequences at the behavioral level.
Bcl-2 interacting protein, BAG-1, binds to and activates the kinase Raf-1.
Authors: Wang H G , Takayama S , Rapp U R , Reed J C
Published: July 2002

The Bcl-2 protein blocks programmed cell death (apoptosis) through an unknown mechanism. Previously we identified a Bcl-2 interacting protein BAG-1 that enhances the anti-apoptotic effects of Bcl-2. Like BAG-1, the serine/threonine protein kinase Raf-1 also can functionally cooperate with Bcl-2 in suppressing apoptosis. Here we show that Raf-1 and BAG-1 specifically interact in vitro and in yeast two-hybrid assays. Raf-1 and BAG-1 can also be coimmunoprecipitated from mammalian cells and from insect cells infected with recombinant baculoviruses encoding these proteins. Furthermore, bacterially-produced BAG-1 protein can increase the kinase activity of Raf-1 in vitro. BAG-1 also activates this mammalian kinase in yeast. These observations suggest that the Bcl-2 binding protein BAG-1 joins Ras and 14-3-3 proteins as potential activators of the kinase Raf-1.
Heterodimer formation and activity in the human enzyme galactose-1-phosphate uridylyltransferase.
Authors: Elsevier J P , Wells L , Quimby B B , Fridovich-Keil J L
Published: July 2002

One of the fundamental questions concerning expression and function of dimeric enzymes involves the impact of naturally occurring mutations on subunit assembly and heterodimer activity. This question is of particular interest for the human enzyme galactose-l-phosphate uridylyl-transferase (GALT), impairment of which results in the inherited metabolic disorder galactosemia, because many if not most patients studied to date are compound heterozygotes rather than true molecular homozygotes. Furthermore, the broad range of phenotypic severity observed in these patients raises the possibility that allelic combination, not just allelic constitution, may play some role in determining outcome. In the work described herein, we have selected two distinct naturally occurring null mutations of GALT, Q188R and R333W, and asked the questions (i) what are the impacts of these mutations on subunit assembly, and (ii) if heterodimers do form, are they active? To answer these questions, we have established a yeast system for the coexpression of epitope-tagged alleles of human GALT and investigated both the extent of specific GALT subunit interactions and the activity of defined heterodimer pools. We have found that both homodimers and heterodimers do form involving each of the mutant subunits tested and that both heterodimer pools retain substantial enzymatic activity. These results are significant not only in terms of their implications for furthering our understanding of galactosemia and GALT holoenzyme structure-function relationships but also because the system described may serve as a model for similar studies of other complexes composed of multiple subunits.
The best thermoelectric.
Authors: Mahan G D , Sofo J O
Published: July 2002

What electronic structure provides the largest figure of merit for thermoelectric materials? To answer that question, we write the electrical conductivity, thermopower, and thermal conductivity as integrals of a single function, the transport distribution. Then we derive the mathematical function for the transport distribution, which gives the largest figure of merit. A delta-shaped transport distribution is found to maximize the thermoelectric properties. This result indicates that a narrow distribution of the energy of the electrons participating in the transport process is needed for maximum thermoelectric efficiency. Some possible realizations of this idea are discussed.
Structure, tissue distribution, and chromosomal localization of the prepronociceptin gene.
Authors: Mollereau C , Simons M J , Soularue P , Liners F , Vassart G , Meunier J C , Parmentier M
Published: July 2002

Nociceptin (orphanin FQ), the newly discovered natural agonist of opioid receptor-like (ORL1) receptor, is a neuropeptide that is endowed with pronociceptive activity in vivo. Nociceptin is derived from a larger precursor, prepronociceptin (PPNOC), whose human, mouse, and rat genes we have now isolated. The PPNOC gene is highly conserved in the three species and displays organizational features that are strikingly similar to those of the genes of preproenkephalin, preprodynorphin, and preproopiomelanocortin, the precursors to endogenous opioid peptides, suggesting the four genes belong to the same family-i.e., have a common evolutionary origin. The PPNOC gene encodes a single copy of nociceptin as well as of other peptides whose sequence is strictly conserved across murine and human species; hence it is likely to be neurophysiologically significant. Northern blot analysis shows that the PPNOC gene is predominantly transcribed in the central nervous system (brain and spinal cord) and, albeit weakly, in the ovary, the sole peripheral organ expressing the gene. By using a radiation hybrid cell line panel, the PPNOC gene was mapped to the short arm of human chromosome 8 (8p21), between sequence-tagged site markers WI-5833 and WI-1172, in close proximity of the locus encoding the neurofilament light chain NEFL. Analysis of yeast artificial chromosome clones belonging to the WC8.4 contig covering the 8p21 region did not allow to detect the presence of the gene on these yeast artificial chromosomes, suggesting a gap in the coverage within this contig.
Single photon emission computerized tomography imaging of amphetamine-induced dopamine release in drug-free schizophrenic subjects.
Authors: Laruelle M , Abi-Dargham A , van Dyck C H , Gil R , D'Souza C D , Erdos J , McCance E , Rosenblatt W , Fingado C , Zoghbi S S , Baldwin R M , Seibyl J P , Krystal J H , Charney D S , Innis R B
Published: July 2002

The dopamine hypothesis of schizophrenia proposes that hyperactivity of dopaminergic transmission is associated with this illness, but direct observation of abnormalities of dopamine function in schizophrenia has remained elusive. We used a newly developed single photon emission computerized tomography method to measure amphetamine-induced dopamine release in the striatum of fifteen patients with schizophrenia and fifteen healthy controls. Amphetamine-induced dopamine release was estimated by the amphetamine-induced reduction in dopamine D2 receptor availability, measured as the binding potential of the specific D2 receptor radiotracer [123I] (S)-(-)-3-iodo-2-hydroxy-6-methoxy-N-[(1-ethyl-2-pyrrolidinyl) methyl]benzamide ([123I]IBZM). The amphetamine-induced decrease in [123I]IBZM binding potential was significantly greater in the schizophrenic group (-19.5 +/- 4.1%) compared with the control group (-7.6 +/- 2.1%). In the schizophrenic group, elevated amphetamine effect on [123I]IBZM binding potential was associated with emergence or worsening of positive psychotic symptoms. This result suggests that psychotic symptoms elicited in this experimental setting in schizophrenic patients are associated with exaggerated stimulation of dopaminergic transmission. Such an observation would be compatible with an abnormal responsiveness of dopaminergic neurons in schizophrenia.
Long-lasting reduction of inhibitory function and gamma-aminobutyric acid type A receptor subunit mRNA expression in a model of temporal lobe epilepsy.
Authors: Rice A , Rafiq A , Shapiro S M , Jakoi E R , Coulter D A , DeLorenzo R J
Published: July 2002

This study evaluated hippocampal inhibitory function and the level of expression of gamma-aminobutyric acid type A (GABAA) receptor mRNA in an in vivo model of epilepsy. Chronic recurrent limbic seizures were induced in rats using injections of pilocarpine. Electrophysiological studies performed on hippocampal slices prepared from control and epileptic animals 1 to 2 months after pilocarpine injections demonstrated a significant hyperexcitability in the epileptic animals. Reduced levels of mRNA expression for the alpha 2 and alpha 5 subunits of the GABAA receptors were evident in the CA1, CA2, and CA3 regions of the hippocampus of epileptic animals. No decrease in mRNA encoding alpha 1, beta 2, or gamma 2 GABAA receptor subunits was observed. In addition, no change in the mRNA levels of alpha CaM kinase II was seen. Selective decreases in mRNA expression did not correlate with neuronal cell loss. The results indicate that selective, long-lasting reduction of GABAA subunit mRNA expression and increased excitability, possibly reflecting loss of GABAergic inhibition, occur in an in vivo model of partial complex epilepsy.
The immunodominant major histocompatibility complex class I-restricted antigen of a murine colon tumor derives from an endogenous retroviral gene product.
Authors: Huang A Y , Gulden P H , Woods A S , Thomas M C , Tong C D , Wang W , Engelhard V H , Pasternack G , Cotter R , Hunt D , Pardoll D M , Jaffee E M
Published: July 2002

Tumors express peptide antigens capable of being recognized by tumor-specific cytotoxic T lymphocytes (CTL). Immunization of mice with a carcinogen-induced colorectal tumor, CT26, engineered to secrete granulocyte/macrophage colony-stimulating factor, routinely generated both short-term and long-term CTL lines that not only lysed the parental tumor in vitro, but also cured mice of established tumor following adoptive transfer in vivo. When either short-term or long-term CTL lines were used to screen peptides isolated from CT26, one reverse-phase high performance liquid chromatography peptide fraction consistently sensitized a surrogate target for specific lysis. The bioactivity remained localized within one fraction following multiple purification procedures, indicating that virtually all of the CT26-specific CTL recognized a single peptide. This result contrasts with other tumor systems, where multiple bioactive peptide fractions have been detected. The bioactive peptide was identified as a nonmutated nonamer derived from the envelope protein (gp70) of an endogenous ecotropic murine leukemia provirus. Adoptive transfer with CTL lines specific for this antigen demonstrated that this epitope represents a potent tumor rejection antigen. The selective expression of this antigen in multiple non-viral-induced tumors provides evidence for a unique class of shared immunodominant tumor associated antigens as targets for antitumor immunity.
Subcellular localization of gamma-aminobutyric acid type A receptors is determined by receptor beta subunits.
Authors: Connolly C N , Wooltorton J R , Smart T G , Moss S J
Published: July 2002

gamma-aminobutyric acid type A (GABAA) receptors are the major sites of fast synaptic inhibition in the brain. They are constructed from four subunit classes with multiple members: alpha (1-6), beta (1-4), gamma (1-4), and delta (1). The contribution of subunit diversity in determining receptor subcellular targeting was examined in polarized Madin-Darby canine kidney (MDCK) cells. Significant detection of cell surface homomeric receptor expression by a combination of both immunological and electrophysiological methodologies was only found for the beta 3 subunit. Expression of alpha/beta binary combinations resulted in a nonpolarized distribution for alpha 1 beta 1 complexes, but specific basolateral targeting of both alpha 1 beta 2 and alpha 1 beta 3 complexes. The polarized distribution of these alpha/beta complexes was unaffected by the presence of the gamma 2S subunit. Interestingly, delivery of receptors containing the beta 3 subunit to the basolateral domain occurs via the apical surface. These results show that beta subunits can selectively target GABAA receptors to distinct cellular locations. Changes in the spatial and temporal expression of beta-subunit isoforms may therefore provide a mechanism for relocating GABAA receptor function between distinct neuronal domains. Given the critical role of these receptors in mediating synaptic inhibition, the contribution of different beta subunits in GABAA receptor function, may have implications in neuronal development and for receptor localization/clustering.
Identification of a nonsense mutation in the carboxyl-terminal region of DNA-dependent protein kinase catalytic subunit in the scid mouse.
Authors: Blunt T , Gell D , Fox M , Taccioli G E , Lehmann A R , Jackson S P , Jeggo P A
Published: July 2002

DNA-dependent protein kinase (DNA-PK) consists of a heterodimeric protein (Ku) and a large catalytic subunit (DNA-PKcs). The Ku protein has double-stranded DNA end-binding activity that serves to recruit the complex to DNA ends. Despite having serine/threonine protein kinase activity, DNA-PKcs falls into the phosphatidylinositol 3-kinase superfamily. DNA-PK functions in DNA double-strand break repair and V(D)J recombination, and recent evidence has shown that mouse scid cells are defective in DNA-PKcs. In this study we have cloned the cDNA for the carboxyl-terminal region of DNA-PKcs in rodent cells and identified the existence of two differently spliced products in human cells. We show that DNA-PKcs maps to the same chromosomal region as the mouse scid gene. scid cells contain approximately wild-type levels of DNA-PKcs transcripts, whereas the V-3 cell line, which is also defective in DNA-PKcs, contains very reduced transcript levels. Sequence comparison of the carboxyl-terminal region of scid and wild-type mouse cells enabled us to identify a nonsense mutation within a highly conserved region of the gene in mouse scid cells. This represents a strong candidate for the inactivating mutation in DNA-PKcs in the scid mouse.
Rat hippocampal neurons express genes for both rod retinal and olfactory cyclic nucleotide-gated channels: novel targets for cAMP/cGMP function.
Authors: Kingston P A , Zufall F , Barnstable C J
Published: July 2002

Cyclic nucleotide-gated (CNG) channels are Ca(2+)-permeable, nonspecific cation channels that can be activated through direct interaction with cAMP and/or cGMP. Recent electrophysiological evidence for these channels in cultured hippocampal neurons prompted us to investigate the expression of CNG channel genes in hippocampus. PCR amplification detected the expression of transcripts for subunit 1 of both the rod photoreceptor (RCNGC1) and the olfactory receptor cell (OCNGC1) subtype of CNG channel in adult rat hippocampus. In situ hybridization detected expression of both channel subtypes in most principal neurons, including pyramidal cells of the CA1 through CA3 regions and granule cells of the dentate gyrus. From the hybridization patterns, we conclude that the two genes are colocalized in individual neurons. Comparison of the patterns of expression of type 1 cGMP-dependent protein kinase and the CNG channels suggests that hippocampal neurons can respond to changes in cGMP levels with both rapid changes in CNG channel activity and slower changes induced by phosphorylation. Future models of hippocampal function should include CNG channels and their effects on both electrical responses and intracellular Ca2+ levels.
A stable human-derived packaging cell line for production of high titer retrovirus/vesicular stomatitis virus G pseudotypes.
Authors: Ory D S , Neugeboren B A , Mulligan R C
Published: July 2002

We have generated a human 293-derived retroviral packaging cell line (293GPG) capable of producing high titers of recombinant Moloney murine leukemia virus particles that have incorporated the vesicular stomatitis virus G (VSV-G) protein. To achieve expression of the retroviral gag-pol polyprotein, the precise coding sequences for gag-pol were introduced into a vector which utilizes totally nonretroviral signals for gene expression. Because constitutive expression of the VSV-G protein is toxic in 293 cells, we used the tetR/VP 16 transactivator and teto minimal promoter system for inducible, tetracycline-regulatable expression of VSV-G. After stable transfection of the 293GPG packaging cell line with the MFG.SnlsLacZ retroviral vector construct, it was possible to readily isolate stable virus-producing cell lines with titers approaching 10(7) colony-forming units/ml. Transient transfection of 293GPG cells using a modified version of MFG.SnlsLacZ, in which the cytomegalovirus IE promoter was used to drive transcription of the proviral genome, led to titers of approximately 10(6) colony-forming units/ml. The retroviral/VSV-G pseudotypes generated using 293GPG cells were significantly more resistant to human complement than commonly used amphotropic vectors and could be highly concentrated (> 1000-fold). This new packaging cell line may prove to be particularly useful for assessing the potential use of retroviral vectors for direct in vivo gene transfer. The design of the cell line also provides at least theoretical advantages over existing cell lines with regard to the possible release of replication-competent virus.
Whole body nitric oxide synthesis in healthy men determined from [15N] arginine-to-[15N]citrulline labeling.
Authors: Castillo L , Beaumier L , Ajami A M , Young V R
Published: July 2002

The rates of whole body nitric oxide (NO) synthesis, plasma arginine flux, and de novo arginine synthesis and their relationships to urea production, were examined in a total of seven healthy adults receiving an L-amino acid diet for 6 days. NO synthesis was estimated by the rate of conversion of the [15N] guanidino nitrogen of arginine to plasma [15N] ureido citrulline and compared with that based on urinary nitrite (NO2-)/nitrate (NO3-) excretion. Six subjects received on dietary day 7, a 24-hr (12-hr fed/12-hr fasted) primed, constant, intravenous infusion of L-[guanidino-15N2]arginine and [13C]urea. A similar investigation was repeated with three of these subjects, plus an additional subject, in which they received L-[ureido-13C]citrulline, to determine plasma citrulline fluxes. The estimated rates (mean +/- SD) of NO synthesis over a period of 24 hr averaged 0.96 +/- 0.1 mumol .kg-1.hr-1 and 0.95 +/- 0.1 mumol.kg-1.hr-1, for the [15N]citrulline and the nitrite/nitrate methods, respectively. About 15% of the plasma arginine turnover was associated with urea formation and 1.2% with NO formation. De novo arginine synthesis averaged 9.2 +/- 1.4 mumol. kg-1.hr-1, indicating that approximately 11% of the plasma arginine flux originates via conversion of plasma citrulline to arginine. Thus, the fraction of the plasma arginine flux associated with NO and also urea synthesis in healthy humans is small, although the plasma arginine compartment serves as a significant precursor pool (54%) for whole body NO formation. This tracer model should be useful for exploring these metabolic relationships in vivo, under specific pathophysiologic states where the L-arginine-NO pathway might be altered.
Oxytocin is required for nursing but is not essential for parturition or reproductive behavior.
Authors: Nishimori K , Young L J , Guo Q , Wang Z , Insel T R , Matzuk M M
Published: July 2002

Oxytocin, a neurohypophyseal hormone, has been traditionally considered essential for mammalian reproduction. In addition to uterine contractions during labor and milk ejection during nursing, oxytocin has been implicated in anterior pituitary function, paracrine effects in the testis and ovary and the neural control of maternal and sexual behaviors. To determine the essential role(s) of oxytocin in mammalian reproductive function, mice deficient in oxytocin have been generated using embryonic stem cell technology. A deletion of exon 1 encoding the oxytocin peptide was generated in embryonic stem cells at a high frequency and was successfully transmitted in the germ line. Southern blot analysis of genomic DNA from homozygote offspring and in situ hybridization with an exonic probe 3' of the deletion failed to detect any oxytocin or neurophysin sequences, respectively, confirming that the mutation was a null mutation. Mice lacking oxytocin are both viable and fertile. Males do not have any reproductive behavioral or functional defects in the absence of oxytocin. Similarly, females lacking oxytocin have no obvious deficits in fertility or reproduction, including gestation and parturition. However, although oxytocin-deficient females demonstrate normal maternal behavior, all offspring die shortly after birth because of the dam's inability to nurse. Postpartum injections of oxytocin to the oxytocin deficient mothers restore milk ejection and rescue the offspring. Thus, despite the multiple reproductive activities that have been attributed to oxytocin, oxytocin plays an essential role only in milk ejection in the mouse.
Hydrogen peroxide-mediated neuronal cell death induced by an endogenous neurotoxin, 3-hydroxykynurenine.
Authors: Okuda S , Nishiyama N , Saito H , Katsuki H
Published: July 2002

3-Hydroxykynurenine (3-HK) is a tryptophan metabolite whose level in the brain is markedly elevated under several pathological conditions, including Huntington disease and human immunodeficiency virus infection. Here we demonstrate that micromolar concentrations (1-100 microM) of 3-HK cause cell death in primary neuronal cultures prepared from rat striatum. The neurotoxicity of 3-HK was blocked by catalase and desferrioxamine but not by superoxide dismutase, indicating that the generation of hydrogen peroxide and hydroxyl radical is involved in the toxicity. Measurement of peroxide levels revealed that 3-HK caused intracellular accumulation of peroxide, which was largely attenuated by application of catalase. The peroxide accumulation and cell death caused by 1-10 microM 3-HK were also blocked by pretreatment with allopurinol or oxypurinol, suggesting that endogenous xanthine oxidase activity is involved in exacerbation of 3-HK neurotoxicity. Furthermore, NADPH diaphorase-containing neurons were spared from toxicity of these concentrations of 3-HK, a finding reminiscent of the pathological characteristics of several neurodegenerative disorders such as Huntington disease. These results suggest that 3-HK at pathologically relevant concentrations renders neuronal cells subject to oxidative stress leading to cell death, and therefore that this endogenous compound should be regarded as an important factor in pathogenesis of neurodegenerative disorders.
Circadian clock-controlled genes isolated from
Neurospora crassa
are late night- to early morning-specific
Authors: Bell-Pedersen Deborah , Shinohara Mari L. , Loros Jennifer J. , Dunlap Jay C.
Published: July 2002

An endogenous circadian biological clock controls the
temporal aspects of life in most organisms, including rhythmic control
of genes involved in clock output pathways. In the fungus
Neurospora crassa
, one pathway known to be under control
of the clock is asexual spore (conidia) development. To understand more
fully the processes that are regulated by the
N. crassa
circadian clock, systematic screens were carried out for genes that
oscillate at the transcriptional level. Time-of-day-specific cDNA
libraries were generated and used in differential screens to identify
six new clock-controlled genes (
ccg
s). Transcripts
specific for each of the
ccg
s preferentially accumulate
during the late night to early morning, although they vary with respect
to steady-state mRNA levels and amplitude of the rhythm. Sequencing of
the ends of the new
ccg
cDNAs revealed that
ccg-12
is identical to
N. crassa cmt
encoding copper metallothionein, providing the suggestion that
not all clock-regulated genes in
N. crassa
are
specifically involved in the development of conidia. This was supported
by finding that half of the new
ccg
s, including
cmt
(
ccg-12
), are not transcriptionally
induced by developmental or light signals. These data suggest a major
role for the clock in the regulation of biological processes distinct
from development.
Conserved circadian elements in phylogenetically diverse algae
Authors: Mittag Maria
Published: July 2002

Circadian expression of the luciferin-binding protein (LBP) from
the dinoflagellate
Gonyaulax polyedra
is regulated at
the translational level. A small interval in the
lbp
3′-untranslated region, which contains seven UG-repeats, serves as a
cis-acting element to which a trans-acting factor (CCTR) binds in a
circadian manner. Its binding activity correlates negatively with the
circadian expression of LBP. Here I report the identification of a
protein in the green alga
Chlamydomonas reinhardtii
that
represents a CCTR analog. It binds both specifically and under control
of the circadian clock to the UG-repeat region. The data show for the
first time that circadian cis-elements implicated in translational
regulation have been conserved during evolution.
Cellular responses to psychomotor stimulant and neuroleptic drugs are abnormal in mice lacking the D1 dopamine receptor
Authors: Moratalla Rosario , Xu Ming , Tonegawa Susumu , Graybiel Ann M.
Published: July 2002

Stimulation of dopamine D1 receptors has profound effects on
addictive behavior, movement control, and working memory. Many of these
functions depend on dopaminergic systems in the striatum and D1–D2
dopamine receptor synergies have been implicated as well. We show here
that deletion of the D1 dopamine receptor produces a neural
phenotype in which amphetamine and cocaine, two addictive
psychomotor stimulants, can no longer stimulate neurons in the striatum
to express cFos or JunB or to regulate dynorphin. By contrast,
haloperidol, a typical neuroleptic that acts preferentially at D2-class
receptors, remains effective in inducing catalepsy and striatal
Fos/Jun expression in the D1 mutants, and these behavioral and
neural effects can be blocked by D2 dopamine receptor agonists. These
findings demonstrate that D2 dopamine receptors can function without
the enabling role of D1 receptors but that D1 dopamine receptors are
essential for the control of gene expression and motor behavior by
psychomotor stimulants.
Results of MDR-1 vector modification trial indicate that granulocyte/macrophage colony-forming unit cells do not contribute to posttransplant hematopoietic recovery following intensive systemic therapy
Authors: Hanania E. G. , Giles R. E. , Kavanagh J. , Ellerson D. , Zu Z. , Wang T. , Su Y. , Kudelka A. , Rahman Z. , Holmes F. , Hortobagyi G. , Claxton D. , Bachier C. , Thall P. , Cheng S. , Hester J. , Ostrove J. M. , Bird R. E. , Chang A. , Korbling M. , Seong D. , Cote R. , Holzmayer T. , Mechetner E. , Heimfeld S. , Berenson R. , Burtness B. , Edwards C. , Bast R. , Andreeff M. , Champlin R. , Deisseroth A. B.
Published: July 2002

To formally test the hypothesis that the
granulocyte/macrophage colony-forming unit (GM-CFU) cells can
contribute to early hematopoietic reconstitution immediately after
transplant, the frequency of genetically modified GM-CFU after
retroviral vector transduction was measured by a quantitative
in
situ
polymerase chain reaction (PCR), which is specific for the
multidrug resistance-1 (MDR-1) vector, and by a quantitative GM-CFU
methylcellulose plating assay. The results of this analysis showed no
difference between the transduction frequency in the products of two
different transduction protocols: “suspension transduction” and
“stromal growth factor transduction.” However, when an analysis
of the frequency of cells positive for the retroviral MDR-1 vector
posttransplantation was carried out, 0 of 10 patients transplanted with
cells transduced by the suspension method were positive for the vector
MDR-1 posttransplant, whereas 5 of 8 patients transplanted with the
cells transduced by the stromal growth factor method were positive for
the MDR-1 vector transcription unit by
in situ
or in
solution PCR assay (a difference that is significant at the
P
= 0.0065 level by the Fisher exact test). These
data suggest that only very small subsets of the GM-CFU fraction of
myeloid cells, if any, contribute to the repopulation of the
hematopoietic tissues that occurs following intensive systemic therapy
and transplantation of autologous hematopoietic cells.
Regeneration of broken tip links and restoration of mechanical transduction in hair cells
Authors: Zhao Yi-dong , Yamoah Ebenezer N. , Gillespie Peter G.
Published: July 2002

A hair cell’s tip links are thought to gate mechanoelectrical
transduction channels. The susceptibility of tip links to acoustic
trauma raises questions as to whether these fragile structures can be
regenerated. We broke tip links with the calcium chelator
1,2-bis(
O
-aminophenoxy)ethane-
N
,
N
,
N
′,
N
′-tetraacetic
acid and found that they can regenerate, albeit imperfectly, over
several hours. The time course of tip-link regeneration suggests that
this process may underlie recovery from temporary threshold shifts
induced by noise exposure. Cycloheximide does not block tip-link
regeneration, indicating that new protein synthesis is not required.
The calcium ionophore ionomycin prevents regeneration, suggesting
regeneration normally may be stimulated by the reduction in
stereociliary Ca
2+
when gating springs rupture and
transduction channels close. Supporting the equivalence of tip links
with gating springs, mechanoelectrical transduction returns over the
same time period as tip links; strikingly, adaptation is substantially
reduced, even 24 hr after breaking tip links.
Hybrid restriction enzymes: zinc finger fusions to Fok I cleavage domain.
Authors: Kim Y G , Cha J , Chandrasegaran S
Published: July 2002

A long-term goal in the field of restriction-modification enzymes has been to generate restriction endonucleases with novel sequence specificities by mutating or engineering existing enzymes. This will avoid the increasingly arduous task of extensive screening of bacteria and other microorganisms for new enzymes. Here, we report the deliberate creation of novel site-specific endonucleases by linking two different zinc finger proteins to the cleavage domain of Fok I endonuclease. Both fusion proteins are active and under optimal conditions cleave DNA in a sequence-specific manner. Thus, the modular structure of Fok I endonuclease and the zinc finger motifs makes it possible to create "artificial" nucleases that will cut DNA near a predetermined site. This opens the way to generate many new enzymes with tailor-made sequence specificities desirable for various applications.
Major histocompatibility complex class I molecule serves as a ligand for presentation of the superantigen staphylococcal enterotoxin B to T cells.
Authors: Häffner A C , Zepter K , Elmets C A
Published: July 2002

Superantigens, such as staphylococcal enterotoxin B (SEB), elicit a strong proliferative response in T cells when presented in the context of major histocompatibility complex (MHC) class II molecules. We observed a similar T-cell response, when MHC class II-negative epidermal cell lines were employed as antigen-presenting cells. Immunoprecipitation studies indicated that the ligand to which SEB bound had a molecular mass of 46 kDa. Radiolabeled SEB could be immunoprecipitated from isolated membrane proteins on the SCC13 epidermal cell line with a monoclonal antibody directed against the MHC class I molecule, and transfection of the K-562 cell line with MHC class I molecules showed a 75% increased SEB-binding capacity compared with the nontransfected MHC class I- and class II-negative counterpart. In functional studies, antibodies to the MHC class I molecule inhibited T-cell proliferation by at least 50%. From these studies, we conclude that MHC class I molecules on malignant squamous cell carcinomas serve as ligands for SEB, which, given the appropriate costimulatory signals, is sufficient to allow for superantigen-induced T-cell proliferation.
Chronic stress alters the immune response to influenza virus vaccine in older adults.
Authors: Kiecolt-Glaser J K , Glaser R , Gravenstein S , Malarkey W B , Sheridan J
Published: July 2002

To determine whether a chronic stressor (caregiving for a spouse with a progressive dementia) is associated with an impaired immune response to influenza virus vaccination, we compared 32 caregivers' vaccine responses with those of 32 sex-, age-, and socioeconomically matched control subjects. Caregivers showed a poorer antibody response following vaccination relative to control subjects as assessed by two independent methods, ELISA and hemagglutination inhibition. Caregivers also had lower levels of in vitro virus-specific-induced interleukin 2 levels and interleukin 1beta; interleukin 6 did not differ between groups. These data demonstrate that down-regulation of the immune response to influenza virus vaccination is associated with a chronic stressor in the elderly. These results could have implications for vulnerability to infection among older adults.
Mimotope/anti-mimotope probing of structural relationships in platelet glycoprotein Ib alpha.
Authors: Miller J L , Lyle V A
Published: July 2002

A bacteriophage library displaying random decapeptides was used to characterize the binding preference of C-34, a monoclonal antibody originally raised against platelet-type von Willebrand disease platelets heterozygous for the mutation 23OWKQ (G --> V)233V234 in the alpha chain of glycoprotein Ib (GPIb alpha). Three rounds of biopanning C-34 against the library resulted in striking convergence upon the sequence WNWRYREYV. Since no portion of this sequence corresponds to a recognizable peptide sequence within human platelet GPIb alpha, it may be considered a "mimotope" of the naturally occurring C-34 epitope, presumably bearing similarity to it in three-dimensional structure. Synthetic AWNWRYREYV peptide preincubated with C-34 fully neutralized the ability of C-34 to inhibit platelet aggregation, with an IC50 of approximately 6 microg/ml. When biotinylated AWNWRYREYV was subsequently bioparmed against the original decapeptide library, the sole clone demonstrating inhibitory activity above background level in a functional platelet assay displayed the sequence RHVAWWRQGV, and chemically synthesized peptide fully inhibited ristocetin-induced aggregation, with an IC50 of 200-400 microg/ml. Synthesized RHVAWWKQGV peptide exerted only slight inhibition, whereas RHVAWWKQVV peptide showed potent inhibitory activity. Moreover, whereas synthesized wild-type 228YVWKQGVDVK237 GPIb alpha peptide was virtually without inhibitory activity, the 228YVWKQ(G -->V) 233VDVK237 peptide fully inhibited ristocetin-induced aggregation, with an IC50 of approximately 400 microg/ml. These studies raise the possibility of an intramolecular association of peptide regions within GPIb alpha that may play a role in the regulation of von Willebrand factor-dependent platelet aggregation.
Initiation process of earthquakes and its implications for seismic hazard reduction strategy.
Authors: Kanamori H
Published: July 2002

For the average citizen and the public, "earthquake prediction" means "short-term prediction," a prediction of a specific earthquake on a relatively short time scale. Such prediction must specify the time, place, and magnitude of the earthquake in question with sufficiently high reliability. For this type of prediction, one must rely on some short-term precursors. Examinations of strain changes just before large earthquakes suggest that consistent detection of such precursory strain changes cannot be expected. Other precursory phenomena such as foreshocks and nonseismological anomalies do not occur consistently either. Thus, reliable short-term prediction would be very difficult. Although short-term predictions with large uncertainties could be useful for some areas if their social and economic environments can tolerate false alarms, such predictions would be impractical for most modern industrialized cities. A strategy for effective seismic hazard reduction is to take full advantage of the recent technical advancements in seismology, computers, and communication. In highly industrialized communities, rapid earthquake information is critically important for emergency services agencies, utilities, communications, financial companies, and media to make quick reports and damage estimates and to determine where emergency response is most needed. Long-term forecast, or prognosis, of earthquakes is important for development of realistic building codes, retrofitting existing structures, and land-use planning, but the distinction between short-term and long-term predictions needs to be clearly communicated to the public to avoid misunderstanding.
Nonuniformity of the constitutive law parameters for shear rupture and quasistatic nucleation to dynamic rupture: a physical model of earthquake generation processes.
Authors: Ohnaka M
Published: July 2002

Based on the recent high-resolution laboratory experiments on propagating shear rupture, the constitutive law that governs shear rupture processes is discussed in view of the physical principles and constraints, and a specific constitutive law is proposed for shear rupture. It is demonstrated that nonuniform distributions of the constitutive law parameters on the fault are necessary for creating the nucleation process, which consists of two phases: (i) a stable, quasistatic phase, and (ii) the subsequent accelerating phase. Physical models of the breakdown zone and the nucleation zone are presented for shear rupture in the brittle regime. The constitutive law for shear rupture explicitly includes a scaling parameter Dc that enables one to give a common interpretation to both small scale rupture in the laboratory and large scale rupture as earthquake source in the Earth. Both the breakdown zone size Xc and the nucleation zone size L are prescribed and scaled by Dc, which in turn is prescribed by a characteristic length lambda c representing geometrical irregularities of the fault. The models presented here make it possible to understand the earthquake generation process from nucleation to unstable, dynamic rupture propagation in terms of physics. Since the nucleation process itself is an immediate earthquake precursor, deep understanding of the nucleation process in terms of physics is crucial for the short-term (or immediate) earthquake prediction.
Bond orientational order, molecular motion, and free energy of high-density DNA mesophases.
Authors: Podgornik R , Strey H H , Gawrisch K , Rau D C , Rupprecht A , Parsegian V A
Published: July 2002

By equilibrating condensed DNA arrays against reservoirs of known osmotic stress and examining them with several structural probes, it has been possible to achieve a detailed thermodynamic and structural characterization of the change between two distinct regions on the liquid-crystalline phase diagram: (i) a higher density hexagonally packed region with long-range bond orientational order in the plane perpendicular to the average molecular direction and (ii) a lower density cholesteric region with fluid-like positional order. X-ray scattering on highly ordered DNA arrays at high density and with the helical axis oriented parallel to the incoming beam showed a sixfold azimuthal modulation of the first-order diffraction peak that reflects the macroscopic bond-orientational order. Transition to the less-dense cholesteric phase through osmotically controlled swelling shows the loss of this bond orientational order, which had been expected from the change in optical birefringence patterns and which is consistent with a rapid onset of molecular positional disorder. This change in order was previously inferred from intermolecular force measurements and is now confirmed by 31P NMR. Controlled reversible swelling and compaction under osmotic stress, spanning a range of densities between approximately 120 mg/ml to approximately 600 mg/ml, allow measurement of the free-energy changes throughout each phase and at the phase transition, essential information for theories of liquid-crystalline states.
Neural mechanisms underlying binocular fusion and stereopsis: Position vs. phase
Authors: Anzai Akiyuki , Ohzawa Izumi , Freeman Ralph D.
Published: July 2002

The visual system utilizes binocular disparity to discriminate the relative depth of objects in space. Since the striate cortex is the first site along the central visual pathways at which signals from the left and right eyes converge onto a single neuron, encoding of binocular disparity is thought to begin in this region. There are two possible mechanisms for encoding binocular disparity through simple cells in the striate cortex: a difference in receptive field (RF) position between the two eyes (RF position disparity) and a difference in RF profile between the two eyes (RF phase disparity). Although there have been studies supporting each of the two encoding mechanisms, both mechanisms have not been examined in a single study. Therefore, the relative roles of the two mechanisms have not been determined. To address this issue, we have mapped left and right eye RFs of simple cells in the cat’s striate cortex using binary m-sequence noise, and then we have estimated RF position and phase disparities. We find that RF position disparities are generally limited to small values that are not sufficient to encode large binocular disparities. In contrast, RF phase disparities cover a wide range of binocular disparities and exhibit dependencies on orientation and spatial frequency in a manner expected for a mechanism that encodes binocular disparity. These results indicate that binocular disparity is mainly encoded through RF phase disparity. However, RF position disparity may play a significant role for cells with high spatial frequency selectivity, which are constrained to small RF phase disparities.
Covalent modification of the active site threonine of proteasomal β subunits and theEscherichia colihomolog HslV by a new class of inhibitors
Authors: Bogyo Matthew , McMaster John S. , Gaczynska Maria , Tortorella Domenico , Goldberg Alfred L. , Ploegh Hidde
Published: July 2002

The proteasome is a multicatalytic protease complex that plays a key role in diverse cellular functions. The peptide vinyl sulfone, carboxybenzyl-leucyl-leucyl-leucine vinyl sulfone (Z-L3VS) covalently inhibits the trypsin-like, chymotrypsin-like and, unlike lactacystin, also the peptidylglutamyl peptidase activity in isolated proteasomes, and blocks their function in living cells. Although described as a class of mechanism-based inhibitors for cysteine proteases, the peptide vinyl sulfone Z-L3VS and a125I-labeled nitrophenol derivative (125I-NIP-L3VS) covalently modify the active site threonine of the catalytic β subunits of the proteasome. Modification of Thermoplasma proteasomes demonstrates the requirement for a hydroxyl amino acid (threonine, serine) as nucleophile at the β subunit’s NH2terminus.125I-NIP-L3VS covalently modifies the HslV subunit of theEscherichia coliprotease complex HslV/HslU, a reaction that requires ATP, and supports a catalytic mechanism shared with that of the eukaryotic proteasome.
enod40
induces dedifferentiation and division of root cortical cells in legumes
Authors: Charon Celine , Johansson Christina , Kondorosi Eva , Kondorosi Adam , Crespi Martin
Published: July 2002

Under nitrogen-limiting conditions
Rhizobium meliloti
can establish symbiosis with
Medicago
plants to form nitrogen-fixing root nodules. Nodule organogenesis starts with the dedifferentiation and division of root cortical cells. In these cells the early nodulin gene
enod40
, which encodes an unusually small peptide (12 or 13 amino acids), is induced from the beginning of this process. Herein we show that
enod40
expression evokes root nodule initiation. (
i
) Nitrogen-deprived transgenic
Medicago truncatula
plants overexpressing
enod40
exhibit extensive cortical cell division in their roots in the absence of
Rhizobium
. (
ii
) Bombardment of
Medicago
roots with an
enod40
-expressing DNA cassette induces dedifferentiation and division of cortical cells and the expression of another early nodulin gene,
Msenod12A
. Moreover, transient expression of either the
enod40
region spanning the oligopeptide sequence or only the downstream region without this sequence induces these responses. Our results suggest that the cell-specific growth response elicited by
enod40
is involved in the initiation of root nodule organogenesis.
Pattern of neuronal activity associated with conscious and unconscious processing of visual signals
Authors: Sahraie A. , Weiskrantz L. , Barbur J. L. , Simmons A. , Williams S. C. R. , Brammer M. J.
Published: July 2002

Following striate cortex damage in monkeys and humans there can be residual function mediated by parallel visual pathways. In humans this can sometimes be associated with a “feeling” that something has happened, especially with rapid movement or abrupt onset. For less transient events, discriminative performance may still be well above chance even when the subject reports no conscious awareness of the stimulus. In a previous study we examined parameters that yield good residual visual performance in the “blind” hemifield of a subject with unilateral damage to the primary visual cortex. With appropriate parameters we demonstrated good discriminative performance, both with and without conscious awareness of a visual event. These observations raise the possibility of imaging the brain activity generated in the “aware” and the “unaware” modes, with matched levels of discrimination performance, and hence of revealing patterns of brain activation associated with visual awareness. The intact hemifield also allows a comparison with normal vision. Here we report the results of a functional magnetic resonance imaging study on the same subject carried out under aware and unaware stimulus conditions. The results point to a shift in the pattern of activity from neocortex in the aware mode, to subcortical structures in the unaware mode. In the aware mode prestriate and dorsolateral prefrontal cortices (area 46) are active. In the unaware mode the superior colliculus is active, together with medial and orbital prefrontal cortical sites.