bluuebunny/crossref_metadata_2025 huggingface dataset:
Progressive decline in avoidance learning paralleled by inflammatory neurodegeneration in transgenic mice expressing interleukin 6 in the brain
Authors: Heyser Charles J. , Masliah Eliezer , Samimi Ana , Campbell Iain L. , Gold Lisa H.
Published: July 2002

Inflammation with expression of interleukin 6 (IL-6) in the brain occurs in many neurodegenerative disorders. To better understand the role of IL-6 in such disorders, we examined performance in a learning task in conjunction with molecular and cellular neuropathology in transgenic mice that express IL-6 chronically from astrocytes in the brain. Transgenic mice exhibited dose- and age-related deficits in avoidance learning that closely corresponded with specific progressive neuropathological changes. These results establish a link between the central nervous system expression of IL-6, inflammatory neurodegeneration, and a learning impairment in transgenic mice. They suggest a critical role for a proinflammatory cytokine in the cognitive deficits and associated neuroinflammatory changes that have been documented in neurodegenerative diseases such as Alzheimer disease and AIDS.
KvLQT1, a voltage-gated potassium channel responsible for human cardiac arrhythmias
Authors: Yang Wen-Pin , Levesque Paul C. , Little Wayne A. , Conder Mary Lee , Shalaby Fouad Y. , Blanar Michael A.
Published: July 2002

The clinical features of long QT syndrome result from episodic life-threatening cardiac arrhythmias, specifically the polymorphic ventricular tachycardia torsades de pointes.
KVLQT1
has been established as the human chromosome 11-linked gene responsible for more than 50% of inherited long QT syndrome. Here we describe the cloning of a full-length
KVLQT1
cDNA and its functional expression.
KVLQT1
encodes a 676-amino acid polypeptide with structural characteristics similar to voltage-gated potassium channels. Expression of KvLQT1 in
Xenopus
oocytes and in human embryonic kidney cells elicits a rapidly activating, K
+
-selective outward current. The I
Kr
-specific blockers, E-4031 and dofetilide, do not inhibit KvLQT1, whereas clofilium, a class III antiarrhythmic agent with the propensity to induce torsades de pointes, substantially inhibits the current. Elevation of cAMP levels in oocytes nearly doubles the amplitude of KvLQT1 currents. Coexpression of minK with KvLQT1 results in a conductance with pharmacological and biophysical properties more similar to I
Ks
than other known delayed rectifier K
+
currents in the heart.
A protein factor that inhibits ATP-dependent glutamate and γ-aminobutyric acid accumulation into synaptic vesicles: Purification and initial characterization
Authors: Özkan Eric D. , Lee Francis S. , Ueda Tetsufumi
Published: July 2002

Glutamate, the major excitatory neurotransmitter in the mammalian central nervous system, is transported into and stored in synaptic vesicles. We have purified to apparent homogeneity a protein from brain cytosol that inhibits glutamate and γ-aminobutyric acid uptake into synaptic vesicles and have termed this protein “inhibitory protein factor” (IPF). IPF refers to three distinct proteins with relative molecular weights of 138,000 (IPF α), 135,000 (IPF β), and 132,000 (IPF γ), respectively. Gel filtration and sedimentation data suggest that all three proteins share an elongated structure, identical Stokes radius (60 Å), and identical sedimentation coefficient (4.3 S). Using these values and a partial specific volume of 0.716 ml/g, we determined the native molecular weight for IPF α to be 103,000. Partial sequence analysis shows that IPF α is derived from α fodrin, a protein implicated in several diverse cellular activities. IPF α inhibits ATP-dependent glutamate uptake into purified synaptic vesicles with an IC
50
of ≈26 nM, while showing no ability to inhibit ATP-independent uptake at concentrations up to 100 nM. Moreover, IPF α inhibited neither norepinephrine uptake into chromaffin vesicles nor Na
+
-dependent glutamate uptake into synaptosomes. However, IPF α inhibited uptake of γ-aminobutyric acid into synaptic vesicles derived from spinal cord, suggesting that inhibition may not be limited to glutamatergic systems. We propose that IPF could be a novel component of a presynaptic regulatory system. Such a system might modulate neurotransmitter accumulation into synaptic vesicles and thus regulate the overall efficacy of neurotransmission.
The hypocretins: Hypothalamus-specific peptides with neuroexcitatory activity
Authors: de Lecea L. , Kilduff T. S. , Peyron C. , Gao X.-B. , Foye P. E. , Danielson P. E. , Fukuhara C. , Battenberg E. L. F. , Gautvik V. T. , Bartlett F. S. , Frankel W. N. , van den Pol A. N. , Bloom F. E. , Gautvik K. M. , Sutcliffe J. G.
Published: July 2002

We describe a hypothalamus-specific mRNA that encodes preprohypocretin, the putative precursor of a pair of peptides that share substantial amino acid identities with the gut hormone secretin. The hypocretin (Hcrt) protein products are restricted to neuronal cell bodies of the dorsal and lateral hypothalamic areas. The fibers of these neurons are widespread throughout the posterior hypothalamus and project to multiple targets in other areas, including brainstem and thalamus. Hcrt immunoreactivity is associated with large granular vesicles at synapses. One of the Hcrt peptides was excitatory when applied to cultured, synaptically coupled hypothalamic neurons, but not hippocampal neurons. These observations suggest that the hypocretins function within the CNS as neurotransmitters.
SMART, a simple modular architecture research tool: Identification of signaling domains
Authors: Schultz Jörg , Milpetz Frank , Bork Peer , Ponting Chris P.
Published: July 2002

Accurate multiple alignments of 86 domains that occur in signaling
proteins have been constructed and used to provide a Web-based tool
(SMART: simple modular architecture research tool) that allows rapid
identification and annotation of signaling domain sequences. The
majority of signaling proteins are multidomain in character with a
considerable variety of domain combinations known. Comparison with
established databases showed that 25% of our domain set could not be
deduced from SwissProt and 41% could not be annotated by Pfam. SMART
is able to determine the modular architectures of single sequences or
genomes; application to the entire yeast genome revealed that at least
6.7% of its genes contain one or more signaling domains, approximately
350 greater than previously annotated. The process of constructing
SMART predicted (
i
) novel domain homologues in
unexpected locations such as band 4.1-homologous domains in focal
adhesion kinases; (
ii
) previously unknown domain
families, including a citron-homology domain; (
iii
)
putative functions of domain families after identification of
additional family members, for example, a ubiquitin-binding role for
ubiquitin-associated domains (UBA); (
iv
) cellular roles
for proteins, such predicted DEATH domains in netrin receptors further
implicating these molecules in axonal guidance; (
v
)
signaling domains in known disease genes such as SPRY domains in both
marenostrin/pyrin and Midline 1; (
vi
) domains in
unexpected phylogenetic contexts such as diacylglycerol
kinase homologues in yeast and bacteria; and (
vii
)
likely protein misclassifications exemplified by a predicted pleckstrin
homology domain in a
Candida albicans
protein,
previously described as an integrin.
Vitamin B
2
-based blue-light photoreceptors in the retinohypothalamic tract as the photoactive pigments for setting the circadian clock in mammals
Authors: Miyamoto Yasuhide , Sancar Aziz
Published: July 2002

In mammals the retina contains photoactive molecules responsible for both vision and circadian photoresponse systems. Opsins, which are located in rods and cones, are the pigments for vision but it is not known whether they play a role in circadian regulation. A subset of retinal ganglion cells with direct projections to the suprachiasmatic nucleus (SCN) are at the origin of the retinohypothalamic tract that transmits the light signal to the master circadian clock in the SCN. However, the ganglion cells are not known to contain rhodopsin or other opsins that may function as photoreceptors. We have found that the two blue-light photoreceptors, cryptochromes 1 and 2 (CRY1 and CRY2), recently discovered in mammals are specifically expressed in the ganglion cell and inner nuclear layers of the mouse retina. In addition, CRY1 is expressed at high level in the SCN and oscillates in this tissue in a circadian manner. These data, in conjunction with the established role of CRY2 in photoperiodism in plants, lead us to propose that mammals have a vitamin A-based photopigment (opsin) for vision and a vitamin B
2
-based pigment (cryptochrome) for entrainment of the circadian clock.
Automated high resolution optical mapping using arrayed, fluid-fixed DNA molecules
Authors: Jing Junping , Reed Jason , Huang John , Hu Xinghua , Clarke Virginia , Edington Joanne , Housman Dan , Anantharaman Thomas S. , Huff Edward J. , Mishra Bud , Porter Brett , Shenker Alexander , Wolfson Estarose , Hiort Catharina , Kantor Ron , Aston Christopher , Schwartz David C.
Published: July 2002

New mapping approaches construct ordered restriction maps from fluorescence microscope images of individual, endonuclease-digested DNA molecules. In optical mapping, molecules are elongated and fixed onto derivatized glass surfaces, preserving biochemical accessibility and fragment order after enzymatic digestion. Measurements of relative fluorescence intensity and apparent length determine the sizes of restriction fragments, enabling ordered map construction without electrophoretic analysis. The optical mapping system reported here is based on our physical characterization of an effect using fluid flows developed within tiny, evaporating droplets to elongate and fix DNA molecules onto derivatized surfaces. Such evaporation-driven molecular fixation produces well elongated molecules accessible to restriction endonucleases, and notably, DNA polymerase I. We then developed the robotic means to grid DNA spots in well defined arrays that are digested and analyzed in parallel. To effectively harness this effect for high-throughput genome mapping, we developed: (
i
) machine vision and automatic image acquisition techniques to work with fixed, digested molecules within gridded samples, and (
ii
) Bayesian inference approaches that are used to analyze machine vision data, automatically producing high-resolution restriction maps from images of individual DNA molecules. The aggregate significance of this work is the development of an integrated system for mapping small insert clones allowing biochemical data obtained from engineered ensembles of individual molecules to be automatically accumulated and analyzed for map construction. These approaches are sufficiently general for varied biochemical analyses of individual molecules using statistically meaningful population sizes.
Atypical antipsychotic drugs selectively increase neurotensin efflux in dopamine terminal regions
Authors: Radke James M. , Owens Michael J. , Ritchie James C. , Nemeroff Charles B.
Published: July 2002

Typical antipsychotic drugs, such as haloperidol and chlorpromazine, increase synthesis of the neuropeptide neurotensin (NT) in both the striatum and the nucleus accumbens, whereas atypical antipsychotic drugs, such as clozapine and olanzapine, do so only in the nucleus accumbens. By using
in vivo
microdialysis, we now report that acute administration of haloperidol, clozapine, or olanzapine failed to alter the release of NT in either the striatum or nucleus accumbens. In contrast, chronic administration of haloperidol for 21 days increased NT release in both the striatum and nucleus accumbens, whereas treatment for 21 days with the atypical antipsychotic drugs, clozapine or olanzapine, increased NT release selectively in the nucleus accumbens. These findings suggest that (
i
) increased NT mRNA expression and NT tissue concentrations are associated with increases in the extracellular fluid concentrations of the peptide and (
ii
) atypical antipsychotic drugs may exert their therapeutic effects and produce fewer side effects by virtue of their selectivity in limbic compared with striatal, target neurons.
Lipid patches in membrane protein oligomers: Crystal structure of the bacteriorhodopsin-lipid complex
Authors: Essen L.-O. , Siegert R. , Lehmann W. D. , Oesterhelt D.
Published: July 2002

Heterogenous nucleation on small molecule crystals causes a monoclinic crystal form of bacteriorhodopsin (BR) in which trimers of this membrane protein pack differently than in native purple membranes. Analysis of single crystals by nano-electrospray ionization-mass spectrometry demonstrated a preservation of the purple membrane lipid composition in these BR crystals. The 2.9-Å x-ray structure shows a lipid-mediated stabilization of BR trimers where the glycolipid S-TGA-1 binds into the central compartment of BR trimers. The BR trimer/lipid complex provides an example of local membrane thinning as the lipid head-group boundary of the central lipid patch is shifted by 5 Å toward the membrane center. Nonbiased electron density maps reveal structural differences to previously reported BR structures, especially for the cytosolic EF loop and the proton exit pathway. The terminal proton release complex now comprises an E194-E204 dyad as a diffuse proton buffer.
Carbohydrate-deficient glycoprotein syndrome type V: Deficiency of dolichyl-
P
-Glc:Man
9
GlcNAc
2
-
PP
-dolichyl glucosyltransferase
Authors: Körner Christian , Knauer Roland , Holzbach Ulrike , Hanefeld Folker , Lehle Ludwig , von Figura Kurt
Published: July 2002

Deficiency of dolichyl-
P
-Glc:Man
9
GlcNAc
2
-
PP
-dolichyl glucosyltransferase is the cause of an additional type of carbohydrate-deficient glycoprotein syndrome (CDGS type V). Clinically this type resembles the classical type Ia of CDGS caused by the deficiency of phosphomannomutase. As a result of the glucosyltransferase deficiency in CDGS type V nonglucosylated lipid-linked oligosaccharides accumulate. The defect is leaky and glucosylated oligosaccharides are found on nascent glycoproteins. The limited availability of glucosylated lipid-linked oligosaccharides explains the incomplete usage of N
-
glycosylation sites in glycoproteins. This finding is reflected in the presence of transferrin forms in serum that lack one or both of the two N
-
linked oligosaccharides and the reduction of mannose incorporation to about one-third of control in glycoproteins of fibroblasts.
Separate jasmonate-dependent and salicylate-dependent defense-response pathways in
Arabidopsis
are essential for resistance to distinct microbial pathogens
Authors: Thomma Bart P. H. J. , Eggermont Kristel , Penninckx Iris A. M. A. , Mauch-Mani Brigitte , Vogelsang Ralph , Cammue Bruno P. A. , Broekaert Willem F.
Published: July 2002

The endogenous plant hormones salicylic acid (SA) and jasmonic acid (JA), whose levels increase on pathogen infection, activate separate sets of genes encoding antimicrobial proteins in
Arabidopsis thaliana
. The pathogen-inducible genes
PR-1
,
PR-2
, and
PR-5
require SA signaling for activation, whereas the plant defensin gene
PDF1.2
, along with a
PR-3
and
PR-4
gene, are induced by pathogens via an SA-independent and JA-dependent pathway. An
Arabidopsis
mutant,
coi1
, that is affected in the JA-response pathway shows enhanced susceptibility to infection by the fungal pathogens
Alternaria brassicicola
and
Botrytis cinerea
but not to
Peronospora parasitica
, and
vice versa
for two
Arabidopsis
genotypes (
npr1
and
NahG
) with a defect in their SA response. Resistance to
P. parasitica
was boosted by external application of the SA-mimicking compound 2,6-dichloroisonicotinic acid [Delaney, T.,
et al
. (1994) Science 266, 1247–1250] but not by methyl jasmonate (MeJA), whereas treatment with MeJA but not 2,6-dichloroisonicotinic acid elevated resistance to
Alternaria brassicicola
. The protective effect of MeJA against
A. brassicicola
was the result of an endogenous defense response activated
in planta
and not a direct effect of MeJA on the pathogen, as no protection to
A. brassicicola
was observed in the
coi1
mutant treated with MeJA. These data point to the existence of at least two separate hormone-dependent defense pathways in
Arabidopsis
that contribute to resistance against distinct microbial pathogens.
Structural invariance of constitutively active and inactive mutants of
Acanthamoeba
myosin IC bound to F-actin in the rigor and ADP-bound states
Authors: Carragher Bridget O. , Cheng Naiqian , Wang Zhen-Yuan , Korn Edward D. , Reilein Amy , Belnap David M. , Hammer John A. , Steven Alasdair C.
Published: July 2002

The three single-headed monomeric myosin I isozymes of
Acanthamoeba castellanii
(AMIs)—AMIA, AMIB, and AMIC—are among the best-studied of all myosins. We have used AMIC to study structural correlates of myosin’s actin-activated ATPase. This activity is normally controlled by phosphorylation of Ser-329, but AMIC may be switched into constitutively active or inactive states by substituting this residue with Glu or Ala, respectively. To determine whether activation status is reflected in structural differences in the mode of attachment of myosin to actin, these mutant myosins were bound to actin filaments in the absence of nucleotide (rigor state) and visualized at 24-Å resolution by using cryoelectron microscopy and image reconstruction. No such difference was observed. Consequently, we suggest that regulation may be affected not by altering the static (time-averaged) structure of AMIC but by modulating its dynamic properties, i.e., molecular breathing. The tail domain of vertebrate intestinal brush-border myosin I has been observed to swing through 31° on binding of ADP. However, it was predicted on grounds of differing kinetics that any such effects with AMIC should be small [Jontes, J. D., Ostap, E. M., Pollard, T. D. & Milligan, R. A. (1998)
J. Cell Biol.
141, 155–162]. We have confirmed this hypothesis by observing actin-associated AMIC in its ADP-bound state. Finally, we compared AMIC to brush-border myosin I and AMIB, which were previously studied under similar conditions. In each case, the shape and angle of attachment to F-actin of the catalytic domain is largely conserved, but the domain structure and disposition of the tail is distinctively different for each myosin.
Using rare mutations to estimate population divergence times: A maximum likelihood approach
Authors: Bertorelle Giorgio , Rannala Bruce
Published: July 2002

In this paper we propose a method to estimate by maximum likelihood the divergence time between two populations, specifically designed for the analysis of nonrecurrent rare mutations. Given the rapidly growing amount of data, rare disease mutations affecting humans seem the most suitable candidates for this method. The estimator
RD
, and its conditional version
RDc
, were derived, assuming that the population dynamics of rare alleles can be described by using a birth–death process approximation and that each mutation arose before the split of a common ancestral population into the two diverging populations. The
RD
estimator seems more suitable for large sample sizes and few alleles, whose age can be approximated, whereas the
RDc
estimator appears preferable when this is not the case. When applied to three cystic fibrosis mutations, the estimator
RD
could not exclude a very recent time of divergence among three Mediterranean populations. On the other hand, the divergence time between these populations and the Danish population was estimated to be, on the average, 4,500 or 15,000 years, assuming or not a selective advantage for cystic fibrosis carriers, respectively. Confidence intervals are large, however, and can probably be reduced only by analyzing more alleles or loci.
Cloning of the cDNA encoding the urotensin II precursor in frog and human reveals intense expression of the urotensin II gene in motoneurons of the spinal cord
Authors: Coulouarn Yolaine , Lihrmann Isabelle , Jegou Sylvie , Anouar Youssef , Tostivint Herve , Beauvillain Jean Claude , Conlon J. Michael , Bern Howard A. , Vaudry Hubert
Published: July 2002

Urotensin II (UII) is a cyclic peptide initially isolated from the caudal neurosecretory system of teleost fish. Subsequently, UII has been characterized from a frog brain extract, indicating that a gene encoding a UII precursor is also present in the genome of a tetrapod. Here, we report the characterization of the cDNAs encoding frog and human UII precursors and the localization of the corresponding mRNAs. In both frog and human, the UII sequence is located at the C-terminal position of the precursor. Human UII is composed of only 11 amino acid residues, while fish and frog UII possess 12 and 13 amino acid residues, respectively. The cyclic region of UII, which is responsible for the biological activity of the peptide, has been fully conserved from fish to human. Northern blot and dot blot analysis revealed that UII precursor mRNAs are found predominantly in the frog and human spinal cord.
In situ
hybridization studies showed that the UII precursor gene is actively expressed in motoneurons. The present study demonstrates that UII, which has long been regarded as a peptide exclusively produced by the urophysis of teleost fish, is actually present in the brain of amphibians and mammals. The fact that evolutionary pressure has acted to conserve fully the biologically active sequence of UII suggests that the peptide may exert important physiological functions in humans.
The acquisition of skilled motor performance: Fast and slow experience-driven changes in primary motor cortex
Authors: Karni Avi , Meyer Gundela , Rey-Hipolito Christine , Jezzard Peter , Adams Michelle M. , Turner Robert , Ungerleider Leslie G.
Published: July 2002

Behavioral and neurophysiological studies suggest that skill
learning can be mediated by discrete, experience-driven changes within
specific neural representations subserving the performance of the
trained task. We have shown that a few minutes of daily practice on a
sequential finger opposition task induced large, incremental
performance gains over a few weeks of training. These gains did not
generalize to the contralateral hand nor to a matched sequence of
identical component movements, suggesting that a lateralized
representation of the learned sequence of movements evolved through
practice. This interpretation was supported by functional MRI data
showing that a more extensive representation of the trained sequence
emerged in primary motor cortex after 3 weeks of training. The imaging
data, however, also indicated important changes occurring in primary
motor cortex during the initial scanning sessions, which we proposed
may reflect the setting up of a task-specific motor processing routine.
Here we provide behavioral and functional MRI data on
experience-dependent changes induced by a limited amount of repetitions
within the first imaging session. We show that this limited training
experience can be sufficient to trigger performance gains that require
time to become evident. We propose that skilled motor performance is
acquired in several stages: “fast” learning, an initial,
within-session improvement phase, followed by a period of consolidation
of several hours duration, and then “slow” learning, consisting of
delayed, incremental gains in performance emerging after continued
practice. This time course may reflect basic mechanisms of neuronal
plasticity in the adult brain that subserve the acquisition and
retention of many different skills.
Neuroimaging studies of word reading
Authors: Fiez Julie A. , Petersen Steven E.
Published: July 2002

This review discusses how neuroimaging can contribute to our
understanding of a fundamental aspect of skilled reading: the ability
to pronounce a visually presented word. One contribution of
neuroimaging is that it provides a tool for localizing brain regions
that are active during word reading. To assess the extent to which
similar results are obtained across studies, a quantitative review of
nine neuroimaging investigations of word reading was conducted. Across
these studies, the results converge to reveal a set of areas active
during word reading, including left-lateralized regions in occipital
and occipitotemporal cortex, the left frontal operculum, bilateral
regions within the cerebellum, primary motor cortex, and the superior
and middle temporal cortex, and medial regions in the supplementary
motor area and anterior cingulate. Beyond localization, the challenge
is to use neuroimaging as a tool for understanding how reading is
accomplished. Central to this challenge will be the integration of
neuroimaging results with information from other methodologies. To
illustrate this point, this review will highlight the importance of
spelling-to-sound consistency in the transformation from orthographic
(word form) to phonological (word sound) representations, and then
explore results from three neuroimaging studies in which the
spelling-to-sound consistency of the stimuli was deliberately varied.
Emphasis is placed on the pattern of activation observed within the
left frontal cortex, because the results provide an example of the
issues and benefits involved in relating neuroimaging results to
behavioral results in normal and brain damaged subjects, and to
theoretical models of reading.
Phosphate availability affects the thylakoid lipid composition and the expression of
SQD1
, a gene required for sulfolipid biosynthesis in
Arabidopsis thaliana
Authors: Essigmann Bernd , Güler Sinan , Narang Ram Avtar , Linke Dirk , Benning Christoph
Published: July 2002

Photosynthetic membranes of higher plants contain specific nonphosphorous lipids like the sulfolipid sulfoquinovosyl diacylglycerol in addition to the ubiquitous phospholipid phosphatidylglycerol. In bacteria, an environmental factor that drastically affects thylakoid lipid composition appears to be the availability of phosphate. Accordingly, we discovered an increase in the relative amount of sulfolipid and a concomitant decrease in phosphatidylglycerol in
Arabidopsis thaliana
grown on medium with reduced amounts of phosphate, as well as in the
pho1
mutant of
A. thaliana
deficient in phosphate transport. To investigate the molecular basis of the observed change in lipid composition, we isolated a cDNA of
A. thaliana
, designated
SQD1
, that encodes a protein involved in sulfolipid biosynthesis as suggested by three lines of evidence. First, the cDNA shows high sequence similarity to bacterial
sqdB
genes known to be essential for sulfolipid biosynthesis; second, the
SQD1
gene product is imported into chloroplasts where sulfolipid biosynthesis takes place; and third, transgenic plants expressing
SQD1
in antisense orientation show a reduction in sulfolipid content. In the
pho1
mutant as well as in wild-type plants grown under reduced phosphate availability, increased amounts of
SQD1
mRNA and SQD1 protein are detected, suggesting that the increase in sulfolipid content under phosphate limitation is the result of an increased expression of at least one gene required for sulfolipid biosynthesis in
A. thaliana
. It is suggested that a certain amount of anionic thylakoid lipid is maintained by substituting sulfolipid for phosphatidylglycerol under reduced phosphate availability.
Peroxynitrite decomposition catalysts: Therapeutics for peroxynitrite-mediated pathology
Authors: Salvemini Daniela , Wang Zhi-Qiang , Stern Michael K. , Currie Mark G. , Misko Thomas P.
Published: July 2002

Inflamed tissue is often characterized by the production of NO and superoxide. These radicals react at diffusion-limited rates to form the powerful oxidant peroxynitrite (PN). When protonated, PN decomposes into either nitrate or reactive intermediates capable of mediating tissue damage by oxidation of protein, lipid, and nucleic acid. We recently have identified porphyrin derivatives capable of catalyzing an increase in nitrate formation with a concomitant decrease in the HO·-like and NO
2
·
-like reactivity of PN. Here, we present evidence for the efficacy of these PN decomposition catalysts both
in vitro
and
in vivo
. Cells in culture were protected from exogenously added PN by the catalyst 5,10,15,20-tetrakis(2,4,6-trimethyl-3,5-disulfonatophenyl)porphyrinato iron (III), whereas free iron and the structurally related compound without iron 5,10,15,20-tetrakis(2,4,6-trimethyl-3,5-disulfonatophenyl)porphyrin did not protect. Cytoprotection correlated well with a reduction in the nitrotyrosine content of released cytosolic proteins, a biochemical marker for PN formation. Carrageenan-induced paw edema is a model of acute inflammation in which PN may play a major role. When tested in this system, both 5,10,15,20-tetrakis(2,4,6-trimethyl-3,5-disulfonatophenyl)porphyrinato iron (III) and 5,10,15,20-tetrakis(
N
-methyl-4′-pyridyl)porphyrinato iron (III) caused a dose-dependent reduction in swelling and lactate dehydrogenase release as well as a detectable shift to nitrate formation in paw tissue. In addition, the catalysts did not elevate mean arterial pressure, suggesting a lack of interaction with NO. Taken together, our data provide compelling evidence supporting the therapeutic value of manipulating PN pharmacologically. Thus, PN decomposition catalysts may represent a unique class of anti-inflammatory agents.
Increase in the cerebrospinal fluid content of neurosteroids in patients with unipolar major depression who are receiving fluoxetine or fluvoxamine
Authors: Uzunova V. , Sheline Y. , Davis J. M. , Rasmusson A. , Uzunov D. P. , Costa E. , Guidotti A.
Published: July 2002

We recently reported that fluoxetine or paroxetine, two selective serotonin reuptake inhibitors (SSRIs), when administered to rats, increase the brain content of the neurosteroid 3α-hydroxy-5α-pregnane-20-one (3α5α-ALLO) without altering the brain content of other neurosteroids. ALLO (3α5α and 3α5β isomers) binds with high affinity to various γ-aminobutyric acid (GABA) receptor A subtypes and facilitates the action of GABA at these receptors. We hypothesized that the increase of ALLO brain content induced by treatment with SSRIs could contribute to alleviating the anxiety and dysphoria associated with the symptomatology of major unipolar depression. We measured ALLO content in four cisternal–lumbar fractions of cerebrospinal fluid (CSF) before and 8–10 weeks after treatment with fluoxetine or fluvoxamine in 15 patients with unipolar major depression. The concentration of ALLO (≈40 fmol/ml in each CSF fraction of three control subjects) was about 60% lower in patients with major unipolar depression. However, in the same patients, fluoxetine or fluvoxamine treatment normalized the CSF ALLO content. Moreover, a statistically significant correlation (
r
= 0.58;
P
< 0.023;
n
= 15) existed between symptomatology improvement (Hamilton Rating Scale for Depression scores) and the increase in CSF ALLO after fluoxetine or fluvoxamine treatment. The CSF content of PREG and PROG remained unaltered after treatment and failed to correlate with the SSRI-induced increase of CSF ALLO. The normalization of CSF ALLO content in depressed patients appears to be sufficient to mediate the anxiolytic and antidysphoric actions of fluoxetine or fluvoxamine via its positive allosteric modulation of GABA type A receptors.
Protection against peroxynitrite-induced fibroblast injury and arthritis development by inhibition of poly(ADP-ribose) synthase
Authors: Szabó Csaba , Virág László , Cuzzocrea Salvatore , Scott Gwen S. , Hake Paul , O’Connor Michael P. , Zingarelli Basilia , Salzman Andrew , Kun Ernest
Published: July 2002

Peroxynitrite, a cytotoxic oxidant formed from nitric oxide (NO) and superoxide, induces DNA strand breakage, which activates the nuclear enzyme poly(ADP-ribose) synthase (PARS; EC2.4.2.30). The cellular function of PARS was determined in fibroblast lines from PARS knockout animals (PARS−/−) and corresponding wild-type animals (PARS+/+), with the aid of the lipophilic PARS inhibitor 5-iodo-6-amino-1,2-benzopyrone (INH2BP). We investigated the role of PARS in peroxynitrite-induced fibroblast injuryin vitroand also in the development of arthritisin vivo. Exposure of embryonic fibroblasts from the PARS+/+animals to peroxynitrite caused DNA single-stand breakage and PARS activation and caused an acute suppression of mitochondrial respiration. INH2BP protected the PARS+/+cells against the suppression of mitochondrial respiration in response to peroxynitrite (50–100 μM). Similarly to PARS inhibition with INH2BP, the PARS−/−cells were protected against peroxynitrite-induced injury. The protection against cellular injury by PARS−/−phenotype or INH2BP waned when cells were challenged with higher concentrations of the oxidant. Inhibition of PARS by INH2BP or by PARS−/−phenotype reduced inducible nitric-oxide synthase (iNOS; EC1.14.13.39) mRNA levels and inhibited production of NO in immunostimulated cells. INH2BP had no peroxynitrite scavenging or hydroxyl radical scavenging effects, and it exerted no additional (nonspecific) effects in the PARS−/−cells. In collagen-induced arthritis, significant staining for nitrotyrosine, a marker of peroxynitrite formation, was found in the inflamed joints. Oral treatment with INH2BP (0.5 g/kg, daily), starting at the onset of arthritis (day 25), delayed the development of the clinical signs at days 26–35 and improved histological status in the knee and paw. Our data demonstrate that deletion of PARS by genetic manipulation or pharmacological inhibition of PARS protects against oxidant-induced cellular injuryin vitroand exhibits anti-inflammatory effectsin vivo.
Plant terpenoid synthases: Molecular biology and phylogenetic analysis
Authors: Bohlmann Jörg , Meyer-Gauen Gilbert , Croteau Rodney
Published: July 2002

This review focuses on the monoterpene, sesquiterpene, and diterpene synthases of plant origin that use the corresponding C
10
, C
15
, and C
20
prenyl diphosphates as substrates to generate the enormous diversity of carbon skeletons characteristic of the terpenoid family of natural products. A description of the enzymology and mechanism of terpenoid cyclization is followed by a discussion of molecular cloning and heterologous expression of terpenoid synthases. Sequence relatedness and phylogenetic reconstruction, based on 33 members of the
Tps
gene family, are delineated, and comparison of important structural features of these enzymes is provided. The review concludes with an overview of the organization and regulation of terpenoid metabolism, and of the biotechnological applications of terpenoid synthase genes.
Exon shuffling mimicked in cell culture
Authors: van Rijk Anke A. F. , de Jong Wilfried W. , Bloemendal Hans
Published: July 2002

Undesired side products of DNA transfections are usually discarded. However, here, we show that such products may provide insight into mutational events that are also a major driving force in protein evolution. While studying the small heat-shock protein αA-crystallin, we transfected the hamster αA-crystallin gene into a mouse muscle cell line. One of the stable transfected cell lines expressed, in addition to the expected normal αA- and alternatively spliced αA
ins
-crystallins, two slightly larger, immunologically cross-reacting proteins. These proteins were found to be encoded by a mutant αA-crystallin gene with a large intragenic duplication, arisen by illegitimate recombination at two CCCAT homologies, ≈1.8 kilobases apart in the normal hamster αA-crystallin gene. As a consequence, a tandem-duplicated exon 3 sequence is present in the mature mRNA of this gene, resulting in a 41-residue repeat in the translated proteins. Cells expressing the elongated αA-crystallins have normal growth characteristics and the usual diffuse cytoplasmic distribution of immunoreactive αA-crystallin. Size-exclusion chromatography of cell extracts indicated that the mutant proteins are readily incorporated into the normal large water-soluble αA-crystallin complexes, showing that the insert does not disturb the integrity of these complexes. This viable αA-crystallin mutant thus mimics the origins and effects of exon duplication, which is a common consequence of exon shuffling in mammalian genome evolution.
Where is my arm? The relative role of vision and proprioception in the neuronal representation of limb position
Authors: Graziano Michael S. A.
Published: July 2002

A central problem in motor control, in the representation of space, and in the perception of body schema is how the brain encodes the relative positions of body parts. According to psychophysical studies, this sense of limb position depends heavily on vision. However, almost nothing is currently known about how the brain uses vision to determine or represent the location of the arm or any other body part. The present experiment shows that the position of the arm is represented in the premotor cortex of the monkey (
Macaca fascicularis
) brain by means of a convergence of visual cues and proprioceptive cues onto the same neurons. These neurons respond to the felt position of the arm when the arm is covered from view. They also respond in a similar fashion to the seen position of a false arm.
Purification of serine racemase: Biosynthesis of the neuromodulator
d
-serine
Authors: Wolosker Herman , Sheth Kevin N. , Takahashi Masaaki , Mothet Jean-Pierre , Brady Roscoe O. , Ferris Christopher D. , Snyder Solomon H.
Published: July 2002

High levels of
d
-serine occur in mammalian brain, where it appears to be an endogenous ligand of the glycine site of
N
-methyl-
d
-aspartate receptors. In glial cultures of rat cerebral cortex,
d
-serine is enriched in type II astrocytes and is released upon stimulation with agonists of non-
N
-methyl-
d
-aspartate glutamate receptors. The high levels of
d
-serine in discrete areas of rat brain imply the existence of a biosynthetic pathway. We have purified from rat brain a soluble enzyme that catalyzes the direct racemization of
l
-serine to
d
-serine. Purified serine racemase has a molecular mass of 37 kDa and requires pyridoxal 5′-phosphate for its activity. The enzyme is highly selective toward
l
-serine, failing to racemize any other amino acid tested. Properties such as pH optimum,
K
m
values, and the requirement for pyridoxal phosphate resemble those of bacterial racemases, suggesting that the biosynthetic pathway for
d
-amino acids is conserved from bacteria to mammalian brain.
Antisense-mediated silencing of a gene encoding a major ryegrass pollen allergen
Authors: Bhalla Prem L. , Swoboda Ines , Singh Mohan B.
Published: July 2002

Type 1 allergic reactions, such as hay fever and allergic asthma, triggered by grass pollen allergens are a global health problem that affects ≈20% of the population in cool, temperate climates. Ryegrass is the dominant source of allergens because of its prodigious production of airborne pollen. Lol p 5 is the major allergenic protein of ryegrass pollen, judging from the fact that almost all of the individuals allergic to grass pollen show presence of serum IgE antibodies against this protein. Moreover, nearly two-thirds of the IgE reactivity of ryegrass pollen has been attributed to this protein. Therefore, it can be expected that down-regulation of Lol p 5 production can significantly reduce the allergic potential of ryegrass pollen. Here, we report down-regulation of Lol p 5 with an antisense construct targeted to theLol p 5gene in ryegrass. The expression of antisense RNA was regulated by a pollen-specific promoter. Immunoblot analysis of proteins with allergen-specific antibodies did not detect Lol p 5 in the transgenic pollen. The transgenic pollen showed remarkably reduced allergenicity as reflected by low IgE-binding capacity of pollen extract as compared with that of control pollen. The transgenic ryegrass plants in whichLol p 5gene expression is perturbed showed normal fertile pollen development, indicating that genetic engineering of hypoallergenic grass plants is possible.
In vivo
suppressor mutations correct a murine model of hereditary tyrosinemia type I
Authors: Manning Kara , Al-Dhalimy Muhsen , Finegold Milton , Grompe Markus
Published: July 2002

Hereditary tyrosinemia type I and alkaptonuria are disorders of tyrosine catabolism caused by deficiency of fumarylacetoacetate hydrolase (FAH) and homogentisic acid dioxygenase (HGD), respectively. Tyrosinemia is a severe childhood disease that affects the liver and kidneys, but alkaptonuria is a more benign adult disorder in comparison. Because HGD is upstream of FAH in the tyrosine pathway, mice doubly mutant in both enzymes were found to be protected from the liver and renal damage of tyrosinemia as hypothesized. Mice mutant at the tyrosinemic locus but heterozygous for alkaptonuria spontaneously developed clonal nodules of functionally normal hepatocytes that were able to rescue the livers of some mice with this genotype. This phenotypic rescue was a result of an inactivating mutation of the wild-type homogentisic acid dioxygenase gene, thus presenting an example of an
in vivo
suppressor mutation in a mammalian model.
Casein kinase Iɛ in the Wnt pathway: Regulation of β-catenin function
Authors: Sakanaka Chie , Leong Peng , Xu Licen , Harrison Stephen D. , Williams Lewis T.
Published: July 2002

Wnt and its intracellular effector β-catenin regulate developmental and oncogenic processes. Using expression cloning to identify novel components of the Wnt pathway, we isolated casein kinase Iɛ (CKIɛ). CKIɛ mimicked Wnt in inducing a secondary axis in
Xenopus
, stabilizing β-catenin, and stimulating gene transcription in cells. Inhibition of endogenous CKIɛ by kinase-defective CKIɛ or CKIɛ antisense-oligonucleotides attenuated Wnt signaling. CKIɛ was in a complex with axin and other downstream components of the Wnt pathway, including Dishevelled. CKIɛ appears to be a positive regulator of the pathway and a link between upstream signals and the complexes that regulate β-catenin.
Oncogenic stimulation recruits cyclin-dependent kinase in the cell cycle start in rat fibroblast
Authors: Jinno Shigeki , Hung Shih-Chieh , Yamamoto Hanako , Lin Jie , Nagata Akihisa , Okayama Hiroto
Published: July 2002

The rat fibroblast NRK cells are transformed reversibly by a combination of growth factors. When stimulated with serum, NRK cells rely on cyclin-dependent kinase 4 (Cdk4) for their S phase entry. However, when stimulated with serum containing oncogenic growth factors, they come to rely on either Cdk4 or Cdk6, and their S phase entry cannot be blocked unless both Cdk4 and Cdk6 are immunodepleted. Such change of dependence does not occur in the NRK cell mutants defective in an oncogenic signal pathway and, therefore, deficient in anchorage-independent cell cycle start ability, correlating Cdk6 dependence with this remarkable, cancer-associated phenotype. However, both Cdk4 and Cdk6 are activated upon serum stimulation, and neither the amounts of Cdk6, Cdk4, cyclin D1, and cyclin-dependent kinase inhibitors nor the activities or subcellular localization of Cdk6 and Cdk4 are significantly influenced by oncogenic stimulation. Thus, oncogenic stimulation invokes Cdk6 to participate in a critical step of the cell cycle start in a rat fibroblast, but by a mechanism seemingly unrelated to the regulation of the kinase. Given that many hematopoietic cells employ predominantly Cdk6 for the cell cycle start and perform anchorage-independent growth by nature, our results raise the possibility that the oncogenic stimulation-induced anchorage-independent cell cycle start of NRK is elicited by a mechanism similar to the one used for hematopoietic cell proliferation.
Serine racemase: A glial enzyme synthesizing
d
-serine to regulate glutamate-
N
-methyl-
d
-aspartate neurotransmission
Authors: Wolosker Herman , Blackshaw Seth , Snyder Solomon H.
Published: July 2002

Although
d
amino acids are prominent in bacteria, they generally are thought not to occur in mammals. Recently, high levels of
d
-serine have been found in mammalian brain where it activates glutamate/
N
-methyl-
d
-aspartate receptors by interacting with the “glycine site” of the receptor. Because amino acid racemases are thought to be restricted to bacteria and insects, the origin of
d
-serine in mammals has been puzzling. We now report cloning and expression of serine racemase, an enzyme catalyzing the formation of
d
-serine from
l
-serine. Serine racemase is a protein representing an additional family of pyridoxal-5′ phosphate-dependent enzymes in eukaryotes. The enzyme is enriched in rat brain where it occurs in glial cells that possess high levels of
d
-serine
in vivo
. Occurrence of serine racemase in the brain demonstrates the conservation of
d
-amino acid metabolism in mammals with implications for the regulation of
N
-methyl-
d
-aspartate neurotransmission through glia-neuronal interactions.
The human granulocyte-macrophage colony- stimulating factor gene is autonomously regulatedin vivoby an inducible tissue-specific enhancer
Authors: Cockerill Peter N. , Bert Andrew G. , Roberts Donna , Vadas Mathew A.
Published: July 2002

The granulocyte-macrophage colony-stimulating factor (GM-CSF) gene is part of a cytokine gene cluster and is directly linked to a conserved upstream inducible enhancer. Here we examined thein vitroandin vivofunctions of the human GM-CSF enhancer and found that it was required for the correctly regulated expression of the GM-CSF gene. An inducible DNase I-hypersensitive site appeared within the enhancer in cell types such as T cells, myeloid cells, and endothelial cells that express GM-CSF, but not in nonexpressing cells. In a panel of transfected cells the human GM-CSF enhancer was activated in a tissue-specific manner in parallel with the endogenous gene. Thein vivofunction of the enhancer was examined in a transgenic mouse model that also addressed the issue of whether the GM-CSF locus was correctly regulated in isolation from other segments of the cytokine gene cluster. After correction for copy number the mean level of human GM-CSF expression in splenocytes from 11 lines of transgenic mice containing a 10.5-kb human GM-CSF transgene was indistinguishable from mouse GM-CSF expression (99% ± 56% SD). In contrast, a 9.8-kb transgene lacking just the enhancer had a significantly reduced (P= 0.004) and more variable level of activity (29% ± 89% SD). From these studies we conclude that the GM-CSF enhancer is required for the correct copy number-dependent expression of the human GM-CSF gene and that the GM-CSF gene is regulated independently from DNA elements associated with the closely linked IL-3 gene or other members of the cytokine gene cluster.
BAS1
: A gene regulating brassinosteroid levels and light responsiveness in
Arabidopsis
Authors: Neff Michael M. , Nguyen Serena M. , Malancharuvil Elizabeth J. , Fujioka Shozo , Noguchi Takahiro , Seto Hideharu , Tsubuki Masayoshi , Honda Toshio , Takatsuto Suguru , Yoshida Shigeo , Chory Joanne
Published: July 2002

The
Arabidopsis
bas1-D
mutation suppresses the long hypocotyl phenotype caused by mutations in the photoreceptor phytochrome B (phyB). The adult phenotype of
bas1-D phyB-4
double mutants mimics that of brassinosteroid biosynthetic and response mutants.
bas1-D phyB-4
has reduced levels of brassinosteroids and accumulates 26-hydroxybrassinolide in feeding experiments. The basis for the mutant phenotype is the enhanced expression of a cytochrome P450 (CYP72B1).
bas1-D
suppresses a
phyB
-null allele, but not a
phyA
-null mutation, and partially suppresses a cryptochrome-null mutation. Seedlings with reduced
BAS1
expression are hyperresponsive to brassinosteroids in a light-dependent manner and display reduced sensitivity to light under a variety of conditions. Thus,
BAS1
represents one of the control points between multiple photoreceptor systems and brassinosteroid signal transduction.