bluuebunny/crossref_metadata_2025 huggingface dataset:
Meis1a suppresses differentiation by G-CSF and promotes proliferation by SCF: Potential mechanisms of cooperativity with Hoxa9 in myeloid leukemia
Authors: Calvo Katherine R. , Knoepfler Paul S. , Sykes David B. , Pasillas Martina P. , Kamps Mark P.
Published: July 2002

Hoxa9 and Meis1a are homeodomain transcription factors that heterodimerize on DNA and are down-regulated during normal myeloid differentiation. Hoxa9 and Meis1a cooperate to induce acute myeloid leukemia (AML) in mice, and are coexpressed in human AML. Despite their cooperativity in leukemogenesis, we demonstrated previously that retroviral expression of Hoxa9 alone—in the absence of coexpressed retroviral Meis1 or of expression of endogenous Meis genes—blocks neutrophil and macrophage differentiation of primary myeloid progenitors cultured in granulocyte–macrophage colony-stimulating factor (GM-CSF). Expression of Meis1 alone did not immortalize any factor-dependent marrow progenitor. Because HoxA9-immortalized progenitors still execute granulocytic differentiation in response to granulocyte CSF (G-CSF) and monocyte differentiation in response to macrophage CSF (M-CSF), we tested the possibility that Meis1a cooperates with Hoxa9 by blocking viable differentiation pathways unaffected by Hoxa9 alone. Here we report that Meis1a suppresses G-CSF-induced granulocytic differentiation of Hoxa9-immortalized progenitors, permitting indefinite self-renewal in G-CSF. Meis1a also reprograms Hoxa9-immortalized progenitors to proliferate, rather than die, in response to stem cell factor (SCF) alone. We propose that Meis1a and Hoxa9 are part of a molecular switch that regulates progenitor abundance by suppressing differentiation and maintaining self-renewal in response to different subsets of cytokines during myelopoiesis. The independent differentiation pathways targeted by Hoxa9 and Meis1a prompt a “cooperative differentiation arrest” hypothesis for a subset of leukemia, in which cooperating transcription factor oncoproteins block complementary subsets of differentiation pathways, establishing a more complete differentiation block
in vivo
.
Femtosecond studies of protein–ligand hydrophobic binding and dynamics: Human serum albumin
Authors: Zhong Dongping , Douhal Abderrazzak , Zewail Ahmed H.
Published: July 2002

In this contribution, we report studies of the nature of the
dynamics and hydrophobic binding in protein–ligand complexes of human
serum albumin with 2-(2′-hydroxyphenyl)-4-methyloxazole. With
femtosecond time resolution, we examined the orientational motion of
the ligand, its intrinsic nuclear motions, and the lifetime changes in
the hydrophobic phase. For comparisons, with similar but chemical
nanocavities, we also studied the same ligand in micelles and
cyclodextrins. The hydrophobic interactions in the binding crevice are
much stronger than those observed in cyclodextrins and micelles. The
confined geometry restrains the nonradiative decay and significantly
lengthens the excited-state lifetime. The observed dynamics over the
femtosecond-to-nanosecond time scale indicate that the binding
structure is rigid and the local motions of the ligand are nearly
“frozen” in the protein. Another major finding is the elucidation
of the directed dynamics by the protein. Proton transfer and
intramolecular twisting of 2-(2′-hydroxyphenyl)-4-methyloxazole were
observed to evolve along two routes: one involves the direct stretching
motion in the molecular plane (≈200 fs) and is not sensitive to the
environment; the second, less dominant, is related to the twisting
motion (≈3 ps) of the two heterocyclic rings and drastically slows
down in the protein hydrophobic pocket.
Enzyme-like proteins by computational design
Authors: Bolon Daniel N. , Mayo Stephen L.
Published: July 2002

We report the development and initial experimental validation of a
computational design procedure aimed at generating enzyme-like protein
catalysts called “protozymes.” Our design approach utilizes a
“compute and build” strategy that is based on the
physical/chemical principles governing protein stability and
catalytic mechanism. By using the catalytically inert
108-residue
Escherichia coli
thioredoxin as a scaffold,
the histidine-mediated nucleophilic hydrolysis of
p
-nitrophenyl acetate as a model reaction, and the
ORBIT protein design software to compute sequences,
an active site scan identified two promising catalytic positions and
surrounding active-site mutations required for substrate binding.
Experimentally, both candidate protozymes demonstrated catalytic
activity significantly above background. One of the proteins, PZD2,
displayed “burst” phase kinetics at high substrate
concentrations, consistent with the formation of a stable enzyme
intermediate. The kinetic parameters of PZD2 are comparable to early
catalytic Abs. But, unlike catalytic Ab design, our design procedure is
independent of fold, suggesting a possible mechanism for examining the
relationships between protein fold and the evolvability of protein
function.
The coxsackievirus and adenovirus receptor is a transmembrane component of the tight junction
Authors: Cohen Christopher J. , Shieh Joseph T. C. , Pickles Raymond J. , Okegawa Takatsugu , Hsieh Jer-Tsong , Bergelson Jeffrey M.
Published: July 2002

The coxsackievirus and adenovirus receptor (CAR) mediates viral
attachment and infection, but its physiologic functions have not been
described. In nonpolarized cells, CAR localized to homotypic
intercellular contacts, mediated homotypic cell aggregation, and
recruited the tight junction protein ZO-1 to sites of cell–cell
contact. In polarized epithelial cells, CAR and ZO-1 colocalized to
tight junctions and could be coprecipitated from cell lysates. CAR
expression led to reduced passage of macromolecules and ions across
cell monolayers, and soluble CAR inhibited the formation of functional
tight junctions. Virus entry into polarized epithelium required
disruption of tight junctions. These results indicate that CAR is a
component of the tight junction and of the functional barrier to
paracellular solute movement. Sequestration of CAR in tight junctions
may limit virus infection across epithelial surfaces.
Efficient expression of protein coding genes from the murine U1 small nuclear RNA promoters.
Authors: Bartlett J S , Sethna M , Ramamurthy L , Gowen S A , Samulski R J , Marzluff W F
Published: July 2002

Few promoters are active at high levels in all cells. Of these, the majority encode structural RNAs transcribed by RNA polymerases I or III and are not accessible for the expression of proteins. An exception are the small nuclear RNAs (snRNAs) transcribed by RNA polymerase II. Although snRNA biosynthesis is unique and thought not to be compatible with synthesis of functional mRNA, we have tested these promoters for their ability to express functional mRNAs. We have used the murine U1a and U1b snRNA gene promoters to express the Escherichia coli lacZ gene and the human alpha-globin gene from either episomal or integrated templates by transfection, or infection into a variety of mammalian cell types. Equivalent expression of beta-galactosidase was obtained from < 250 nucleotides of 5'-flanking sequence containing the complete promoter of either U1 snRNA gene or from the 750-nt cytomegalovirus promoter and enhancer regions. The mRNA was accurately initiated at the U1 start site, efficiently spliced and polyadenylylated, and localized to polyribosomes. Recombinant adenovirus containing the U1b-lacZ chimeric gene transduced and expressed beta-galactosidase efficiently in human 293 cells and airway epithelial cells in culture. Viral vectors containing U1 snRNA promoters may be an attractive alternative to vectors containing viral promoters for persistent high-level expression of therapeutic genes or proteins.
Impaired locomotor activity and exploratory behavior in mice lacking histamine H
1
receptors
Authors: Inoue Isao , Yanai Kazuhiko , Kitamura Daisuke , Taniuchi Ichiro , Kobayashi Takashi , Niimura Kaku , Watanabe Takehiko , Watanabe Takeshi
Published: July 2002

From pharmacological studies using histamine antagonists and
agonists, it has been demonstrated that histamine modulates many
physiological functions of the hypothalamus, such as arousal state,
locomotor activity, feeding, and drinking. Three kinds of receptors
(H
1
, H
2
, and H
3
) mediate these
actions. To define the contribution of the histamine H
1
receptors (H1R) to behavior, mutant mice lacking the H1R were generated
by homologous recombination. In brains of homozygous mutant mice, no
specific binding of [
3
H]pyrilamine was seen.
[
3
H]Doxepin has two saturable binding sites with higher
and lower affinities in brains of wild-type mice, but H1R-deficient
mice showed only the weak labeling of [
3
H]doxepin that
corresponds to lower-affinity binding sites. Mutant mice develop
normally, but absence of H1R significantly increased the ratio of
ambulation during the light period to the total ambulation for 24 hr in
an accustomed environment. In addition, mutant mice significantly
reduced exploratory behavior of ambulation and rearings in a new
environment. These results indicate that through H1R, histamine is
involved in circadian rhythm of locomotor activity and exploratory
behavior as a neurotransmitter.
The tumor necrosis factor receptor 2 signal transducers TRAF2 and c-IAP1 are components of the tumor necrosis factor receptor 1 signaling complex
Authors: Shu Hong-Bing , Takeuchi Masahiro , Goeddel David V.
Published: July 2002

The two cell surface receptors for tumor necrosis factor (TNF) interact with a number of intracellular signal transducing proteins. The association of TRADD, a 34-kDa cytoplasmic protein containing a C-terminal death domain, with aggregated TNF receptor 1 (TNF-R1) through their respective death domains leads to NF-κB activation and programmed cell death. In contrast, TNF receptor 2 (TNF-R2) interacts with the TNF receptor associated factors 2/1 (TRAF2/TRAF1) heterocomplex, which mediates the recruitment of two cellular inhibitor of apoptosis proteins (c-IAP1 and c-IAP2) to TNF-R2. Here we show that the TNF-R2 signal transducers TRAF2 and c-IAP1 are a part of the TNF-R1 signaling complex. The recruitment of TRAF2 and c-IAP1 to TNF-R1 is TNF-dependent, is mediated by TRADD, and is independent of TNF-R2. These data establish the physiological involvement of TRAF2 and c-IAP1 in TNF-R1 signaling and help provide a molecular explanation for both the overlapping and distinct signals generated by the two TNF receptors.
Ferredoxin and ferredoxin–heme maquettes
Authors: Gibney Brian R. , Mulholland Stephen E. , Rabanal Francesc , Dutton P. Leslie
Published: July 2002

A 16-amino acid residue peptide derived from a consensus motif of
natural ferredoxins incorporates a tetranuclear iron sulfur cluster
under physiological conditions. Successful assembly of the
[4Fe–4S]
2+/1+
cluster within a monomeric peptide was
demonstrated using size exclusion chromatography, UV-visible, visible
CD, and cryogenic EPR spectroscopies. The robustness of
[4Fe–4S]
2+/1+
formation was tested using peptides with
either the ligating cysteine exchanged for alanine or with the
intervening amino acids replaced by glycine. The small size of the
peptide allows for modular incorporation into more complex protein
structures. In one larger structure, we describe a tetra-α-helix
bundle that self-assembles both iron–sulfur clusters and hemes,
thereby demonstrating feasibility for the general synthesis of
maquettes containing multiple, juxtaposed redox cofactors. This is a
motif common to the catalytic sites of native oxidoreductases.
Definition of the HLA-A29 peptide ligand motif allows prediction of potential T-cell epitopes from the retinal soluble antigen, a candidate autoantigen in birdshot retinopathy.
Authors: Boisgerault F , Khalil I , Tieng V , Connan F , Tabary T , Cohen J H , Choppin J , Charron D , Toubert A
Published: July 2002

The peptide-binding motif of HLA-A29, the predisposing allele for birdshot retinopathy, was determined after acid-elution of endogenous peptides from purified HLA-A29 molecules. Individual and pooled HPLC fractions were sequenced by Edman degradation. Major anchor residues could be defined as glutamate at the second position of the peptide and as tyrosine at the carboxyl terminus. In vitro binding of polyglycine synthetic peptides to purified HLA-A29 molecules also revealed the need for an auxiliary anchor residue at the third position, preferably phenylalanine. By using this motif, we synthesized six peptides from the retinal soluble antigen, a candidate autoantigen in autoimmune uveoretinitis. Their in vitro binding was tested on HLA-A29 and also on HLA-B44 and HLA-B61, two alleles sharing close peptide-binding motifs. Two peptides derived from the carboxyl-terminal sequence of the human retinal soluble antigen bound efficiently to HLA-A29. This study could contribute to the prediction of T-cell epitopes from retinal autoantigens implicated in birdshot retinopathy.
Purified inositol hexakisphosphate kinase is an ATP synthase: diphosphoinositol pentakisphosphate as a high-energy phosphate donor.
Authors: Voglmaier S M , Bembenek M E , Kaplin A I , Dormán G , Olszewski J D , Prestwich G D , Snyder S H
Published: July 2002

Diphosphoinositol pentakisphosphate (PP-IP5) and bis(diphospho)inositol tetrakisphosphate (bis-PP-IP4) are recently identified inositol phosphates that possess pyrophosphate bonds. We have purified an inositol hexakisphosphate (IP6) kinase from rat brain supernatants. The pure protein, a monomer of 54 kDa, displays high affinity (Km = 0.7 microM) and selectivity for inositol hexakisphosphate as substrate. It can be dissociated from bis(diphospho)inositol tetrakisphosphate synthetic activity. The purified enzyme transfers a phosphate from PP-IP5 to ADP to form ATP. This ATP synthase activity indicates the high phosphate group transfer potential of PP-IP5 and may represent a physiological role for PP-IP5.
From oligonucleotide shapes to genomic SELEX: Novel biological regulatory loops
Authors: Gold Larry , Brown David , He Yi-yuan , Shtatland Timur , Singer Britta S. , Wu Yan
Published: July 2002

The SELEX method and oligonucleotide combinatorial chemistry discovery process yields high-affinity/high-specificity ligands for virtually any molecular target. Typically, the enormous starting libraries used in the SELEX process contain 10
14
–10
15
sequences. We now ask if the smaller sequences, complexity of extant organisms, and evolutionary history provide useful interactions between oligonucleotides and at least some unexpected targets. That is, do organisms contain a robust “linkage map” between their oligonucleotides and proteins and/or small molecules that enriches life?
A computer screening approach to immunoglobulin superfamily structures and interactions: Discovery of small non-peptidic CD4 inhibitors as novel immunotherapeutics
Authors: Li Song , Gao Jimin , Satoh Takashi , Friedman Thea M. , Edling Andrea E. , Koch Ute , Choksi Swati , Han Xiaobing , Korngold Robert , Huang Ziwei
Published: July 2002

The interaction between CD4 and major histocompatibility complex (MHC) class II proteins is critical for the activation of CD4+T cells, which are involved in transplantation reactions and a number of autoimmune diseases. In this study we have identified a CD4 surface pocket as a functional epitope implicated in CD4–MHC class II interaction and T-cell activation. A computer-based strategy has been used to screen ≈150,000 non-peptidic organic compounds in a molecular data base and to identify a group of compounds as ligands of the proposed CD4 surface pocket. These small organic compounds have been shown to specifically block stable CD4–MHC class II binding, and exhibit significant inhibition of immune responses in animal models of autoimmune disease and allograft transplant rejection, suggesting their potential as novel immunosuppressants. This structure-based computer screening approach may have general implications for studying many immunoglobulin-like structures and interactions that share similar structural features. Furthermore, the results from this study have demonstrated that the rational design of small non-peptidic inhibitors of large protein–protein interfaces may indeed be an achievable goal.
Localized gene action controlling intestinal neoplasia in mice
Authors: Gould Karen A. , Dove William F.
Published: July 2002

Mice heterozygous for the
Apc

Min

(
Min
) mutation develop adenomas throughout the intestinal tract.
Apc
is believed to be involved in cell migration, adhesion, and polarity. Adenoma multiplicity and growth rate are modulated by an unlinked modifier locus,
Mom1
. The secretory phospholipase
Pla2g2a
is a candidate for
Mom1
. Here, we investigate the range of action of
Apc
and
Mom1
. Analysis of chimeric Min mice indicates that the actions of both
Apc
and
Mom1
are localized within the cell lineage that gives rise to intestinal tumors.
The thioredoxin binding domain of bacteriophage T7 DNA polymerase confers processivity on
Escherichia coli
DNA polymerase I
Authors: Bedford Ella , Tabor Stanley , Richardson Charles C.
Published: July 2002

Bacteriophage T7 DNA polymerase shares extensive sequence homology with
Escherichia coli
DNA polymerase I. However,
in vivo
,
E. coli
DNA polymerase I is involved primarily in the repair of DNA whereas T7 DNA polymerase is responsible for the replication of the viral genome. In accord with these roles, T7 DNA polymerase is highly processive while
E. coli
DNA polymerase I has low processivity. The high processivity of T7 DNA polymerase is achieved through tight binding to its processivity factor,
E. coli
thioredoxin. We have identified a unique 76-residue domain in T7 DNA polymerase responsible for this interaction. Insertion of this domain into the homologous site in
E. coli
DNA polymerase I results in a dramatic increase in the processivity of the chimeric DNA polymerase, a phenomenon that is dependent upon its binding to thioredoxin.
Control of membrane phosphatidylcholine biosynthesis by diacylglycerol levels in neuronal cells undergoing neurite outgrowth
Authors: Araki Wataru , Wurtman Richard J.
Published: July 2002

Phospholipids are the major components of cell membranes and are required for cellular growth. We studied membrane phosphatidylcholine (PtdCho) biosynthesis in neuronal cells undergoing neurite outgrowth, by using PC12 cells as a model system. When neurite outgrowth was induced by exposing PC12 cells to nerve growth factor for 2 and 4 days, the amounts of [
14
C]choline incorporated into [
14
C]phosphatidylcholine per cell (i.e., per DNA) increased approximately 5- and 10-fold, respectively, as compared with control cells, reflecting increases in the rate of PtdCho biosynthesis. [
14
C]choline uptake was not affected. Analysis of the three major PtdCho biosynthetic enzymes showed that the activity of CDPcholine:1,2-diacylglycerol cholinephosphotransferase was increased by approximately 50% after nerve growth factor treatment, but the activities of choline kinase or choline-phosphate cytidylyltransferase were unaltered; the cholinephosphotransferase displayed a high
K
m
value (≈1,200 μM) for diacylglycerol. Moreover, free cellular diacylglycerol levels increased by approximately 1.5- and 4-fold on the second and fourth days, respectively. These data indicate that PtdCho biosynthesis is enhanced when PC12 cells sprout neurites, and the enhancement is mediated primarily by changes in cholinephosphotransferase activity and its saturation with diacylglycerol. This suggests a novel regulatory role for diacylglycerol in membrane phospholipid biosynthesis.
Phalangeal curvature and positional behavior in extinct sloth lemurs (Primates, Palaeopropithecidae)
Authors: Jungers William L. , Godfrey Laurie R. , Simons Elwyn L. , Chatrath Prithijit S.
Published: July 2002

Recent paleontological discoveries in Madagascar document the existence of a diverse clade of palaeopropithecids or “sloth lemurs”:
Mesopropithecus
(three species),
Babakotia
(one species),
Palaeopropithecus
(three species), and
Archaeoindris
(one species). This mini-radiation of now extinct (“subfossil”) lemurs is most closely related to the living indrids (
Indri
,
Propithecus
, and
Avahi
). Whereas the extant indrids are known for their leaping acrobatics, the palaeopropithecids (except perhaps for the poorly known giant
Archaeoindris
) exhibit numerous skeletal design features for antipronograde or suspensory positional behaviors (e.g., high intermembral indices and mobile joints). Here we analyze the curvature of the proximal phalanges of the hands and feet. Computed as the included angle (θ), phalangeal curvature develops in response to mechanical use and is known to be correlated in primates with hand and foot function in different habitats; terrestrial species have straighter phalanges than their arboreal counterparts, and highly suspensory forms such as the orangutan possess the most curved phalanges. Sloth lemurs as a group are characterized by very curved proximal phalanges, exceeding those seen in spider monkeys and siamangs, and approaching that of orangutans. Indrids have curvatures roughly half that of sloth lemurs, and the more terrestrial, subfossil
Archaeolemur
possesses the least curved phalanges of all the indroids. Taken together with many other derived aspects of their postcranial anatomy, phalangeal curvature indicates that the sloth lemurs are one of the most suspensory clades of mammals ever to evolve.
Polyclonal structure of intestinal adenomas in
Apc

Min

/+ mice with concomitant loss of
Apc
+
from all tumor lineages
Authors: Merritt A. J. , Gould K. A. , Dove W. F.
Published: July 2002

When tumors form in intestinal epithelia, it is important to know whether they involve single initiated somatic clones. Advanced carcinomas in humans and mice are known to be monoclonal. However, earlier stages of tumorigenesis may instead involve an interaction between cells that belong to separate somatic clones within the epithelium. The clonality of early tumors has been investigated in mice with an inherited predisposition to intestinal tumors. Analysis of Min (multiple intestinal neoplasia) mice chimeric for a ubiquitously expressed cell lineage marker revealed that normal intestinal crypts are monoclonal, but intestinal adenomas frequently have a polyclonal structure, presenting even when very small as single, focal adenomas composed of at least two somatic lineages. Furthermore, within these polyclonal adenomas, all tumor lineages frequently lose the wild-type
Apc
allele. These observations can be interpreted by several models for clonal interaction within the epithelium, ranging from passive fusion within regions of high neoplastic potential to a requirement for active clonal cooperation.
Sodium channel selectivity filter regulates antiarrhythmic drug binding
Authors: Sunami Akihiko , Dudley Samuel C. , Fozzard Harry A.
Published: July 2002

Local anesthetic antiarrhythmic drugs block Na
+
channels and have important clinical uses. However, the molecular mechanism by which these drugs block the channel has not been established. The family of drugs is characterized by having an ionizable amino group and a hydrophobic tail. We hypothesized that the charged amino group of the drug may interact with charged residues in the channel’s selectivity filter. Mutation of the putative domain III selectivity filter residue of the adult rat skeletal muscle Na
+
channel (μ1) K1237E increased resting lidocaine block, but no change was observed in block by neutral analogs of lidocaine. An intermediate effect on the lidocaine block resulted from K1237S and there was no effect from K1237R, implying an electrostatic effect of Lys. Mutation of the other selectivity residues, D400A (domain I), E755A (domain II), and A1529D (domain IV) allowed block by externally applied quaternary membrane-impermeant derivatives of lidocaine (QX314 and QX222) and accelerated recovery from block by internal QX314. Neo-saxitoxin and tetrodotoxin, which occlude the channel pore, reduced the amount of QX314 bound in D400A and A1529D, respectively. Block by outside QX314 in E755A was inhibited by mutation of residues in transmembrane segment S6 of domain IV that are thought to be part of an internal binding site. The results demonstrate that the Na
+
channel selectivity filter is involved in interactions with the hydrophilic part of the drugs, and it normally limits extracellular access to and escape from their binding site just within the selectivity filter. Participation of the selectivity ring in antiarrhythmic drug binding and access locates this structure adjacent to the S6 segment.
Sequence-specific RNA binding by an SR protein requires RS domain phosphorylation: Creation of an SRp40-specific splicing enhancer
Authors: Tacke Roland , Chen Yan , Manley James L.
Published: July 2002

We showed previously that ASF/SF2, a member of the SR protein family of splicing factors, can activate a splicing enhancer element composed of high-affinity ASF/SF2 binding sites. To determine whether other SR proteins can behave similarly, we selected a high-affinity RNA-binding site (B1) for the SR protein SRp40. Strikingly, the success of this selection was completely dependent on phosphorylation of the RS domain, as unphosphorylated SRp40 failed to select specific sequences. We show that three copies of B1 function as a strong splicing enhancer, activating an intron with suboptimal splicing signals in nuclear extracts. Enhancer activity in S100 extracts (which lack SR proteins) required SRp40 and a nuclear fraction previously found to be required for ASF/SF2-dependent splicing. Importantly, enhancer activity was lost when SRp40 was replaced by ASF/SF2 or SC35, and SRp40 was the only classical SR protein found to be associated with the enhancer. Together, our results indicate that phosphorylation-dependent, sequence-specific RNA binding can impart unique activities to individual SR proteins.
Inhibition of the p44/42 MAP kinase pathway protects hippocampal neurons in a cell-culture model of seizure activity
Authors: Murray Beverley , Alessandrini Alessandro , Cole Andrew J. , Yee Ann G. , Furshpan Edwin J.
Published: July 2002

Excessive release of glutamate and the subsequent influx of calcium are associated with a number of neurological insults that result in neuronal death. The calcium-activated intracellular signaling pathways responsible for this excitotoxic injury are largely unknown. Here, we report that PD098059, a selective inhibitor of the calcium-activated p44/42 mitogen-activated protein kinase (MAP kinase) pathway, reduces neuronal death in a cell-culture model of seizure activity. Dissociated hippocampal neurons grown chronically in the presence of kynurenate, a broad spectrum glutamate-receptor antagonist, and elevated amounts of magnesium exhibit intense seizure-like activity after the removal of these blockers of excitatory synaptic transmission. A 30-min removal of the blockers produced extensive neuronal death within 24 h as assayed by the uptake of trypan blue and the release of lactate dehydrogenase. Phospho-p44/42 MAP kinase immunoreactivity after 30 min of seizure-like activity was present in many neuronal somata and dendrites as well as some synaptic terminals, consistent with both the presynaptic and postsynaptic effects of this pathway. The addition of PD098059 (40 μM; EC
50
= 10 μM) during a 30-min washout of synaptic blockers inhibited the phosphorylation of p44/42 MAP kinase and reduced both the trypan-blue staining (
n
= 13) and the release of lactate dehydrogenase (
n
= 16) by 73% ± 18% and 75% ± 19% (mean ± SD), respectively. The observed neuroprotection could be caused by an effect of PD098059 on seizure-like events or on downstream signaling pathways activated by the seizure-like events. Either possibility suggests a heretofore unknown function for the p44/42 MAP kinase pathway in neurons.
An oculomotor representation area within the ventral premotor cortex
Authors: Fujii Naotaka , Mushiake Hajime , Tanji Jun
Published: July 2002

We explored the ventral part of the premotor cortex (PMV) with intracortical microstimulation (ICMS) while monkeys performed a visual fixation task, to see whether the PMV is involved in oculomotor control. ICMS evoked saccades from a small-restricted region in the PMV, without evoking movements in the limbs, neck, or body. We found the saccade-evoking site in the PMV in a total of three hemispheres in two monkeys. Quantitative analysis of the effects of eye position on saccades evoked by microstimulation of the PMV characterized the evoked saccades as goal directed. The nature of the saccades evoked in the PMV contrasted with the fixed vector nature of saccades evoked by ICMS of the frontal eye field. We also found that neurons in this restricted area of the PMV were active while the animals were performing a saccade task that required them to make saccades toward targets without arm movements. These data provide evidence for the presence of an oculomotor-specific subregion within the PMV. This subregion and the surrounding skeletomotor-representing regions of the PMV seem to coordinate oculomotor and skeletomotor control in performing goal-directed motor tasks.
Analysis of the
Drosophila
host defense in
domino
mutant larvae, which are devoid of hemocytes
Authors: Braun Anne , Hoffmann Jules A. , Meister Marie
Published: July 2002

We have analyzed the
Drosophila
immune response in
domino
mutant larvae, which are devoid of blood cells. The
domino
mutants have a good larval viability, but they die as prepupae. We show that, on immune challenge, induction of the genes encoding antimicrobial peptides in the fat body is not affected significantly in the mutant larvae, indicating that hemocytes are not essential in this process. The hemocoele of
domino
larvae contains numerous live microorganisms, the presence of which induces a weak antimicrobial response in the fat body. A full response is observed only after septic injury. We propose that the fat body cells are activated both by the presence of microorganisms and by injury and that injury potentiates the effect of microorganisms. Survival experiments after an immune challenge showed that
domino
mutants devoid of blood cells maintain a wild-type resistance to septic injury. This resistance was also observed in mutant larvae in which the synthesis of antibacterial peptides is impaired (
immune deficiency
larvae) and in mutants that are deficient for humoral melanization (
Black cells
larvae). However, if
domino
was combined with either the
immune deficiency
or the
Black cell
mutation, the resistance to septic injury was reduced severely. These results establish the relevance of the three immune reactions: phagocytosis, synthesis of antibacterial peptides, and melanization. By working in synergy, they provide
Drosophila
a highly effective defense against injury and/or infection.
Functional analyses of troponin T mutations that cause hypertrophic cardiomyopathy: Insights into disease pathogenesis and troponin function
Authors: Sweeney H. Lee , Feng Huisheng S. , Yang Zhaouhui , Watkins Hugh
Published: July 2002

Mutations in a number of cardiac sarcomeric protein genes cause hypertrophic cardiomyopathy (HCM). Previous findings indicate that HCM-causing mutations associated with a truncated cardiac troponin T (TnT) and missense mutations in the β-myosin heavy chain share abnormalities in common, acting as dominant negative alleles that impair contractile performance. In contrast, Lin
et al
. [Lin, D., Bobkova, A., Homsher, E. & Tobacman, L. S. (1996)
J. Clin. Invest.
97, 2842–2848] characterized a TnT point mutation (Ile79Asn) and concluded that it might lead to hypercontractility and, thus, potentially a different mechanism for HCM pathogenesis. In this study, three HCM-causing cardiac TnT mutations (Ile79Asn, Arg92Gln, and ΔGlu160) were studied in a myotube expression system. Functional studies of wild-type and mutant transfected myotubes revealed that all three mutants decreased the calcium sensitivity of force production and that the two missense mutations (Ile79Asn and Arg92Gln) increased the unloaded shortening velocity nearly 2-fold. The data demonstrate that TnT can alter the rate of myosin cross-bridge detachment, and thus the troponin complex plays a greater role in modulating muscle contractile performance than was recognized previously. Furthermore, these data suggest that these TnT mutations may cause disease via an increased energetic load on the heart. This would represent a second paradigm for HCM pathogenesis.
The variable domain of nonassembled Ig light chains determines both their half-life and binding to the chaperone BiP
Authors: Skowronek Markus H. , Hendershot Linda M. , Haas Ingrid G.
Published: July 2002

Not much is known about the features that determine the biological stability of a molecule retained in the endoplasmic reticulum (ER). Ig light (L) chains that are not secreted in the absence of Ig heavy (H) chain expression bind to the ER chaperone BiP as partially folded molecules until they are degraded. Although all Ig L chains have the same three-dimensional structure when part of an antibody molecule, the degradation rate of unassembled Ig L chains is not identical. For instance, the two nonsecreted murine Ig L chains, κNS1and λFS62, are degraded with half-lives of approximately 1 and 4 hr, respectively, in the same NS1 myeloma cells. Furthermore, the BiP/λFS62Ig L chain complex appears to be more stable than the BiP/κNS1complex. Here, we used the ability of single Ig domains to form an internal disulfide bond after folding as a measure of the folding state of κNS1and λFS62Ig L chains. Both of these nonsecreted L chains lack the internal disulfide bond in the variable (V) domain, whereas the constant (C) domain was folded in that respect. In both cases the unfolded V domain provided the BiP binding site. The stability of BiP binding to these two nonsecreted proteins was quite different, and both the stability of the BiP:Ig L chain complex and the half-life of the Ig L chain could be transferred from one Ig L chain isotype to the other by swapping the V domains. Our data suggest that the physical stability of BiP association with an unfolded region of a given light chain determines the half-life of that light chain, indicating a direct link between chaperone interaction and delivery of partially folded substrates to the mammalian degradation machinery.
The Black–Scholes pricing formula in the quantum context
Authors: Segal Wiliam , Segal I. E.
Published: July 2002

A natural explanation for extreme irregularities in the evolution of prices in financial markets is provided by quantum effects. The lack of simultaneous observability of relevant variables and the interference of attempted observation with the values of these variables represent such effects. These characteristics have been noted by traders and economists and appear intrinsic to market dynamics. This explanation is explored here in terms of a corresponding generalization of the Wiener process and its role in the Black–Scholes–Merton theory. The differentiability of the Wiener process as a sesquilinear form on a dense domain in the Hilbert space of square-integrable functions over Wiener space is shown and is extended to the quantum context. This provides a basis for a corresponding generalization of the Ito theory of stochastic integration. An extension of the Black–Scholes option pricing formula to the quantum context is deduced.
Are protein folds atypical?
Authors: Li Hao , Tang Chao , Wingreen Ned S.
Published: July 2002

Protein structures are a very special class among all possible structures. It has been suggested that a “designability principle” plays a crucial role in nature’s selection of protein sequences and structures. Here, we provide a theoretical base for such a selection principle, using a simple model of protein folding based on hydrophobic interactions. A structure is reduced to a string of 0s and 1s, which represent the surface and core sites, respectively, as the backbone is traced. Each structure is therefore associated with one point in a high dimensional space. Sequences are represented by strings of their hydrophobicities and thus can be mapped into the same space. A sequence that lies closer to a particular structure in this space than to any other structures will have that structure as its ground state. Atypical structures, namely those far away from other structures in the high dimensional space, have more sequences that fold into them and are thermodynamically more stable. We argue that the most common folds of proteins are the most atypical in the space of possible structures.
Modulation of ventricular function through gene transfer
in vivo
Authors: Hajjar Roger J. , Schmidt Ulrich , Matsui Takashi , Guerrero J. Luis , Lee Kyung-Han , Gwathmey Judith K. , Dec G. William , Semigran Marc J. , Rosenzweig Anthony
Published: July 2002

We used a catheter-based technique to achieve generalized cardiac gene transfer
in vivo
and to alter cardiac function by overexpressing phospholamban (PL) which regulates the activity of the sarcoplasmic reticulum Ca
2+
ATPase (SERCA2a). By using this approach, rat hearts were transduced
in vivo
with 5 × 10
9
pfu of recombinant adenoviral vectors carrying cDNA for either PL, β-galactosidase (β-gal), or modified green fluorescent protein (EGFP). Western blot analysis of ventricles obtained from rats transduced by Ad.PL showed a 2.8-fold increase in PL compared with hearts transduced by Ad.βgal. Two days after infection, rat hearts transduced with Ad.PL had lower peak left ventricular pressure (58.3 ± 12.9 mmHg,
n
= 8) compared with uninfected hearts (92.5 ± 3.5 mmHg,
n
= 6) or hearts infected with Ad.βgal (92.6 ± 5.9 mmHg,
n
= 6). Both peak rate of pressure rise and pressure fall (+3, 210 ± 298 mmHg/s, −2, 117 ± 178 mmHg/s,
n
= 8) were decreased in hearts overexpressing PL compared with uninfected hearts (+5, 225 ± 136 mmHg/s, −3, 805 ± 97 mmHg/s,
n
= 6) or hearts infected with Ad.βgal (+5, 108 ± 167 mmHg/s, −3, 765 ± 121 mmHg/s,
n
= 6). The time constant of left ventricular relaxation increased significantly in hearts overexpressing PL (33.4 ± 3.2 ms,
n
= 8) compared with uninfected hearts (18.5 ± 1.0 ms,
n
= 6) or hearts infected with Ad.βgal (20.8 ± 2.1 ms,
n
= 6). These differences in ventricular function were maintained 7 days after infection. These studies open the prospect of using somatic gene transfer to modulate overall cardiac function
in vivo
for either experimental or therapeutic applications.
Use of plant roots for phytoremediation and molecular farming
Authors: Gleba Doloressa , Borisjuk Nikolai V. , Borisjuk Ludmyla G. , Kneer Ralf , Poulev Alexander , Skarzhinskaya Marina , Dushenkov Slavik , Logendra Sithes , Gleba Yuri Y. , Raskin Ilya
Published: July 2002

Alternative agriculture, which expands the uses of plants well
beyond food and fiber, is beginning to change plant biology. Two
plant-based biotechnologies were recently developed that take advantage
of the ability of plant roots to absorb or secrete various substances.
They are (
i
) phytoextraction, the use of plants to
remove pollutants from the environment and (
ii
)
rhizosecretion, a subset of molecular farming, designed to produce and
secrete valuable natural products and recombinant proteins from roots.
Here we discuss recent advances in these technologies and assess their
potential in soil remediation, drug discovery, and molecular farming.
Reversible inactivation of the nucleus basalis magnocellularis induces disruption of cortical acetylcholine release and acquisition, but not retrieval, of aversive memories
Authors: Miranda María Isabel , Bermúdez-Rattoni Federico
Published: July 2002

The basal forebrain complex, which includes the nucleus basalis magnocellularis (NBM), provides widespread cholinergic and γ-aminobutyric acid-containing projections throughout the brain, including the insular and pyriform cortices. A number of studies have implicated the cholinergic neurons in the mediation of learning and memory processes. However, the role of basal forebrain activity in information retrieval mechanisms is less known. The aim of the present study is to evaluate the effects of reversible inactivation of the NBM by tetrodotoxin (TTX, a voltage-sensitive sodium channel blocker) during the acquisition and retrieval of conditioned taste aversion (CTA) and to measure acetylcholine (ACh) release during TTX inactivation in the insular cortex, by means of the microdialysis technique in free-moving rats. Bilateral infusion of TTX in the NBM was performed 30 min before the presentation of gustative stimuli, in either the CTA acquisition trial or retrieval trial. At the same time, levels of extracellular ACh release were measured in the insular cortex. The behavioral results showed significant impairment in CTA acquisition when the TTX was infused in the NBM, whereas retrieval was not affected when the treatment was given during the test trial. Biochemical results showed that TTX infusion into the NBM produced a marked decrease in cortical ACh release as compared with the controls during consumption of saccharin in the acquisition trial. Depleted ACh levels were found during the test trial in all groups except in the group that received TTX during acquisition. These results suggest a cholinergic-dependent process during acquisition, but not during memory retrieval, and that NBM-mediated cholinergic cortical release may play an important role in early stages of learning, but not during recall of aversive memories.
The propagation of binding interactions to remote sites in proteins: Analysis of the binding of the monoclonal antibody D1.3 to lysozyme
Authors: Freire Ernesto
Published: July 2002

The interaction of a ligand with a protein occurs at a local site
(the binding site) and involves only a few residues; however, the
effects of that interaction are often propagated to remote locations.
The chain of events initiated by binding provides the basis for
fundamental biological phenomena such as allosterism, signal
transduction, and structural-stability modification. In this paper, a
structure-based statistical thermodynamic approach is presented and
used to predict the propagation of the stabilization effects triggered
by the binding of the monoclonal antibody D1.3 to hen egg white
lysozyme. Previously, Williams
et al.
[Williams,
D. C., Benjamin, D. C., Poljak, R. J. & Rule, G. S.
(1996)
J. Mol. Biol.
257, 866–876] showed that the
binding of this antibody affects the stability of hen egg white
lysozyme and that the binding effects propagate to a selected number of
residues at remote locations from the binding epitope. In this paper,
we show that this phenomenon can be predicted from structure. The
formalism presented here permits the identification of the structural
path followed by cooperative interactions that originate at the binding
site. It is shown that an important condition for the propagation of
binding effects to distal regions is the presence of a significant
fraction of residues with low structural stability in the uncomplexed
binding site. A survey of protein structures indicates that many
binding sites have a dual character and are defined by regions of high
and low structural stabilities. The low-stability regions might be
involved in the transmission of binding information to other regions in
the protein.
Mice lacking all conventional MHC class II genes
Authors: Madsen Lars , Labrecque Nathalie , Engberg Jan , Dierich Andrée , Svejgaard Arne , Benoist Christophe , Mathis Diane , Fugger Lars
Published: July 2002

MHC class II (MHC-II) molecules play a central role in the selection of the T cell repertoire, in the establishment and regulation of the adaptive immune response, and in autoimmune deviation. We have generated knockout mice lacking all four of the classical murine MHC-II genes (MHCIIΔ/Δmice), via a large (80-kilobase) deletion of the entire class II region that was engineered by homologous recombination and Cre recombinase-mediated excision. These mice feature immune system perturbations like those of Aα and Aβ knockout animals, notably a dearth of CD4+lymphocytes in the thymus and spleen. No new anatomical or physiological abnormalities were observed in MHCIIΔ/Δmice. Because these animals are devoid of all classical MHC-II chains, even unpaired chains, they make excellent recipients for MHC-II transgenes from other species, avoiding the problem of interspecies cross-pairing of MHC-II chains. Therefore, they should be invaluable for engineering “humanized” mouse models of human MHC-II-associated autoimmune disorders.
Solution structure of Apaf-1 CARD and its interaction with caspase-9 CARD: A structural basis for specific adaptor/caspase interaction
Authors: Zhou Pei , Chou James , Olea Roberto Sanchez , Yuan Junying , Wagner Gerhard
Published: July 2002

Direct recruitment and activation of caspase-9 by Apaf-1 through the homophilic CARD/CARD (
Ca
spase
R
ecruitment
D
omain) interaction is critical for the activation of caspases downstream of mitochondrial damage in apoptosis. Here we report the solution structure of the Apaf-1 CARD domain and its surface of interaction with caspase-9 CARD. Apaf-1 CARD consists of six tightly packed amphipathic α-helices and is topologically similar to the RAIDD CARD, with the exception of a kink observed in the middle of the N-terminal helix. By using chemical shift perturbation data, the homophilic interaction was mapped to the acidic surface of Apaf-1 CARD centered around helices 2 and 3. Interestingly, a significant portion of the chemically perturbed residues are hydrophobic, indicating that in addition to the electrostatic interactions predicted previously, hydrophobic interaction is also an important driving force underlying the CARD/CARD interaction. On the basis of the identified functional residues of Apaf-1 CARD and the surface charge complementarity, we propose a model of CARD/CARD interaction between Apaf-1 and caspase-9.
A simple relationship between viral load and survival time in HIV-1 infection
Authors: Arnaout Ramy A. , Lloyd Alun L. , O’Brien Thomas R. , Goedert James J. , Leonard John M. , Nowak Martin A.
Published: July 2002

Despite important recent insights into the short-term dynamics of HIV-1 infection, our understanding of the long-term pathogenesis of AIDS remains unclear. Using an approach that places rapid progressors, typical progressors, and nonprogressors on a single clinical spectrum of disease progression, we quantitate the previously reported relationship between viral load and survival time. We introduce the concept of viral constant, present evidence that this quantity is conserved across patients, and explore the immunopathological implications of this finding. We conclude with a quantitative approach for assessing the benefits of a given regime of antiviral therapy.