bluuebunny/crossref_metadata_2025 huggingface dataset:
Five subunits are required for reconstitution of the cleavage and polyadenylation activities of
Saccharomyces cerevisiae
cleavage factor I
Authors: Gross Stefan , Moore Claire
Published: July 2002

Cleavage and polyadenylation of mRNA 3′ ends in
Saccharomyces cerevisiae
requires several factors, one
of which is cleavage factor I (CF I). Purification of CF I activity
from yeast extract has implicated numerous proteins as functioning in
both cleavage and/or polyadenylation. Through reconstitution of
active CF I from separately expressed and purified proteins, we show
that CF I contains five subunits, Rna14, Rna15, Pcf11, Clp1, and Hrp1.
These five are necessary and sufficient for reconstitution of cleavage
activity
in vitro
when mixed with CF II, and for
specific polyadenylation when mixed with polyadenylation factor I,
purified poly(A) polymerase, and poly(A) binding protein. Analysis of
the individual protein–protein interactions supports an architectural
model for CF I in which Pcf11 simultaneously interacts with Rna14,
Rna15, and Clp1, whereas Rna14 bridges Rna15 and Hrp1.
CTXφ contains a hybrid genome derived from tandemly integrated elements
Authors: Davis Brigid M. , Waldor Matthew K.
Published: July 2002

CTXφ is a filamentous, temperate bacteriophage whose genome includes
ctxAB
, the genes that encode cholera toxin. In toxigenic isolates of
Vibrio cholerae
, tandem arrays of prophage DNA, usually interspersed with the related genetic element RS1, are integrated site-specifically within the chromosome. We have discovered that these arrays routinely yield hybrid virions, composed of DNA from two adjacent prophages or from a prophage and a downstream RS1. Coding sequences are always derived from the 5′ prophage whereas most of an intergenic sequence, intergenic region 1, is always derived from the 3′ element. The presence of tandem elements is required for production of virions:
V. cholerae
strains that contain a solitary prophage rarely yield CTX virions, and the few virions detected result from imprecise excision of prophage DNA. Thus, generation of the replicative form of CTXφ, pCTX, a step that precedes production of virions, does not depend on reversal of the process for site-specific integration of CTXφ DNA into the
V. cholerae
chromosome. Production of pCTX also does not depend on RecA-mediated homologous recombination between adjacent prophages. We hypothesize that the CTXφ-specific proteins required for replication of pCTX can also function on a chromosomal substrate, and that, unlike the processes used by other integrating phages, production of pCTX and CTXφ does not require excision of the prophage from the chromosome. Use of this replication strategy maximizes vertical transmission of prophage DNA while still enabling dissemination of CTXφ to new hosts.
Genes identified by an expression screen of the vector mosquito
Anopheles gambiae
display differential molecular immune response to malaria parasites and bacteria
Authors: Oduol Frederick , Xu Jiannong , Niaré Oumou , Natarajan Ramya , Vernick Kenneth D.
Published: July 2002

We performed a gene expression screen of the entire transcriptome
of the major African malaria vector
Anopheles gambiae
for immune response genes in adult female mosquitoes, which is the
developmental stage infected by malaria parasites. Mosquitoes were
immune-stimulated for subtractive cloning by treatment with bacterial
lipopolysaccharide, a potent and general elicitor of the innate immune
response, and by injury. The screen yielded a highly enriched cDNA
library in which more than half of the clones were immune responsive.
In this paper, we describe 23 immune-regulated genes, including
putative protease inhibitors, serine proteases, regulatory molecules,
and a number of genes without known relatives. A molecule related to
the protease inhibitor α-2-macroglobulin responded strongly to
malaria parasite infection, but displayed little or no response to
bacteria, whereas other genes exhibited the inverse pattern. These
results indicate that the insect immune system discriminates between
molecular signals specific to infection with bacteria and malaria
parasites.
Characterization of the nucleotide-binding capacity and the ATPase activity of the PIP
3
-binding protein JFC1
Authors: Catz Sergio D. , Johnson Jennifer L. , Babior Bernard M.
Published: July 2002

In this work, we demonstrate that the phosphatidylinositol
3,4,5-trisphosphate-binding protein JFC1 is an ATP-binding protein with
magnesium-dependent ATPase activity. We show that JFC1 specifically
binds to the ATP analog 8-azido-[α-
32
P]ATP. The
affinity of JFC1 for [α-
32
P]ATP was 10× greater than
its affinity for [α-
32
P]ADP; the protein did not appear
to bind to [α-
32
P]GTP. JFC1 hydrolyzed
[α-
32
P]ATP in a Mg
2+
-dependent manner.
JFC1, which also hydrolyzed dATP, has a relatively high affinity for
ATP, with a
K
M
value of 58 μM, and a
k
cat
value of 2.27 per min. The predicted
amino acid sequence of JFC1 denotes a putative nucleotide-binding site
similar to those in the GHKL ATPase/kinase superfamily. However, a
truncation of JFC1 that contains boxes G2 and G3 but not boxes N and G1
of the Bergerat-binding site showed residual ATPase activity. Secondly,
the antitumor ATP-mimetic agent geldanamycin, which inhibits the ATPase
activity of Hsp-90, did not affect JFC1 ATPase. Therefore, the
characteristics of the ATP-binding site of JFC1 are unique.
Phosphatidylinositol 3,4,5-trisphosphate, a high-affinity ligand of
JFC1 did not affect its ATPase kinetics parameters, suggesting that the
phosphoinositide have a different role in JFC1 function.
Erratic overdispersion of three molecular clocks: GPDH, SOD, and XDH
Authors: Rodríguez-Trelles Francisco , Tarrío Rosa , Ayala Francisco J.
Published: July 2002

The neutrality theory predicts that the rate of neutral
molecular evolution is constant over time, and thus that there is a
molecular clock for timing evolutionary events. It has been observed
that the variance of the rate of evolution is generally larger than
expected according to the neutrality theory, which has raised the
question of how reliable the molecular clock is or, indeed, whether
there is a molecular clock at all. We have carried out an extensive
investigation of three proteins, glycerol-3-phosphate dehydrogenase
(GPDH), superoxide dismutase (SOD), and xanthine dehydrogenase (XDH).
We have observed that (
i
) the three proteins evolve
erratically through time and across lineages and (
ii
)
the erratic patterns of acceleration and deceleration differ from locus
to locus, so that one locus may evolve faster in one than another
lineage, whereas the opposite may be the case for another locus. The
observations are inconsistent with the predictions made by various
subsidiary hypotheses proposed to account for the overdispersion of the
molecular clock.
An
in vivo
ESR spin-trapping study: Free radical generation in rats from formate intoxication— role of the Fenton reaction
Authors: Dikalova Anna E. , Kadiiska Maria B. , Mason Ronald P.
Published: July 2002

Electron spin resonance spectroscopy has been used to study free radical generation in rats with acute sodium formate poisoning. The
in vivo
spin-trapping technique was used with α-(4-pyridyl-1-oxide)-
N
-
t
-butylnitrone (POBN), which reacts with free radical metabolites to form radical adducts, which were detected in the bile and urine samples from Fischer rats. The use of [
13
C]-sodium formate and computer simulations of the spectra identified the 12-line spectrum as arising from the POBN/carbon dioxide anion radical adduct. The identification of POBN/
⋅
CO

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radical adduct provides direct electron spin resonance spectroscopy evidence for the formation of
⋅
CO

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radicals during acute intoxication by sodium formate, suggesting a free radical metabolic pathway. To study the mechanism of free radical generation by formate, we tested several known inhibitors. Both allopurinol, an inhibitor of xanthine oxidase, and aminobenzotriazole, a cytochrome P450 inhibitor, decreased free radical formation from formate, which may imply a dependence on hydrogen peroxide. In accord with this hypothesis, the catalase inhibitor 3-aminotriazole caused a significant increase in free radical formation. The iron chelator Desferal decreased the formation of free radicals up to 2-fold. Presumably, iron plays a role in the mechanism of free radical generation by formate via the Fenton reaction. The detection of formate free radical metabolites generated
in vivo
and the key role of the Fenton reaction in this process may be important for understanding the pathogenesis of both formate and methanol intoxication.
Prediction of disclinations in nematic elastomers
Authors: Fried Eliot , Todres Russell E.
Published: July 2002

We present a theory for uniaxial nematic elastomers with variable
asphericity. As an application of the theory, we consider the
time-independent, isochoric extension of a right circular cylinder.
Numerical solutions to the resulting differential equation are obtained
for a range of extensions. For sufficiently large extensions, there
exists an isotropic core of material surrounding the cylinder axis
where the asphericity vanishes and in which the polymeric molecules are
shaped as spherical coils. This region, corresponding to a disclination
of strength +1 manifesting itself along the axis, is bounded by a
narrow transition layer across which the asphericity drops rapidly and
attains a nontrivial negative value. Away from the disclination, the
material is anisotropic, and the polymeric molecules are shaped as
ellipsoidal coils of revolution oblate about the radial direction.
Along with the area of steeply changing asphericity between isotropic
and anisotropic regimes, a marked drop in the free-energy density is
observed. The boundary of the disclination core is associated with the
location of this energy drop. For realistic choices of material
parameters, this criterion yields a core on the order of
10
−2
μm, which coincides with observations in
conventional liquid crystal melts. Finally, we find that the total
energy definitively shows a preference for disclinated states.
Integration of complete transferred DNA units is dependent on the activity of virulence E2 protein of Agrobacterium tumefaciens.
Authors: Rossi L , Hohn B , Tinland B
Published: July 2002

Agrobacterium tumefaciens transfers transferred DNA (T-DNA), a single-stranded segment of its tumor-inducing (Ti) plasmid, to the plant cell nucleus. The Ti-plasmid-encoded virulence E2 (VirE2) protein expressed in the bacterium has single-stranded DNA (ssDNA)-binding properties and has been reported to act in the plant cell. This protein is thought to exert its influence on transfer efficiency by coating and accompanying the single-stranded T-DNA (ss-T-DNA) to the plant cell genome. Here, we analyze different putative roles of the VirE2 protein in the plant cell. In the absence of VirE2 protein, mainly truncated versions of the T-DNA are integrated. We infer that VirE2 protects the ss-T-DNA against nucleolytic attack during the transfer process and that it is interacting with the ss-T-DNA on its way to the plant cell nucleus. Furthermore, the VirE2 protein was found not to be involved in directing the ss-T-DNA to the plant cell nucleus in a manner dependent on a nuclear localization signal, a function which is carried by the NLS of VirD2. In addition, the efficiency of T-DNA integration into the plant genome was found to be VirE2 independent. We conclude that the VirE2 protein of A. tumefaciens is required to preserve the integrity of the T-DNA but does not contribute to the efficiency of the integration step per se.
The C-proteinase that processes procollagens to fibrillar collagens is identical to the protein previously identified as bone morphogenic protein-1.
Authors: Li S W , Sieron A L , Fertala A , Hojima Y , Arnold W V , Prockop D J
Published: July 2002

Bone morphogenic protein-1 (BMP-1) was originally identified as one of several BMPs that induced new bone formation when implanted into ectopic sites in rodents. BMP-1, however, differed from other BMPs in that it its structure was not similar to transforming growth factor beta. Instead, it had a large domain homologous to a metalloendopeptidase isolated from crayfish, an epidermal growth-factor-like domain, and three regions of internal sequence homology referred to as CUB domains. Therefore, BMP-1 was a member of the "astacin families" of zinc-requiring endopeptidases. Many astacins have been shown to play critical roles in embryonic hatching, dorsal/ventral patterning, and early developmental decisions. Here, we have obtained amino acid sequences and isolated cDNA clones for procollagen C-proteinase (EC 3.4.24.19), an enzyme that is essential for the processing of procollagens to fibrillar collagens. The results demonstrate that procollagen C-proteinase is identical to BMP-1.
Targeted mutation of Ncam to produce a secreted molecule results in a dominant embryonic lethality.
Authors: Rabinowitz J E , Rutishauser U , Magnuson T
Published: July 2002

The neural cell adhesion molecule (NCAM) is a membrane-associated member of the immunoglobulin superfamily capable of both homophilic and heterophilic binding. To investigate the significance of this binding, a gene targeting strategy in embryonic stem (ES) cells was used to replace the membrane-associated forms of NCAM with a soluble, secreted form of its extracellular domain. Although the heterozygous mutant ES cells were able to generate low coat color chimeric mice, only the wild-type allele was transmitted, suggesting the possibility of dominant lethality. Analysis of chimeric embryos with high level of ES cell contribution revealed severe growth retardation and morphological defects by E8.5-E9.5. The second allele was also targeted, and embryos derived almost entirely from the homozygous mutant ES cells exhibited the same lethal phenotype as observed with heterozygous chimeras. Together, these results indicate that dominant lethality associated with the secreted NCAM does not require the presence of membrane-associated NCAM. Furthermore, the data indicate that potent bioactive cues or signals can be generated by NCAM.
The stress response to ionizing radiation involoves c-Abl-dependent phosphorylation of SHPTP1.
Authors: Kharbanda S , Bharti A , Pei D , Wang J , Pandey P , Ren R , Weichselbaum R , Walsh C T , Kufe D
Published: July 2002

c-Abl is a nonreceptor tyrosine kinase that is activated by certain DNA-damaging agents. The present studies demonstrate that nuclear c-Abl binds constitutively to the protein tyrosine phosphatase SHPTP1. Treatment with ionizing radiation is associated with c-Abl-dependent tyrosine phosphorylation of SHPTP1. The results demonstrate that the SH3 domain of c-Abl interacts with a WPDHGVPSEP motif (residues 417-426) in the catalytic domain of SHPTP1 and that c-Abl phosphorylates C terminal Y536 and Y564 sites. The functional significance of the c-Abl-SHPTP1 interaction is supported by the demonstration that, like c-Abl, SHPTP1 regulates the induction of Jun kinase activity following DNA damage. These findings indicate that SHPTP1 is involved in the response to genotoxic stress through a c-Abl-dependent mechanism.
Evolutionary changes in the expression pattern of a developmentally essential gene in three Drosophila species.
Authors: Wang D , Marsh J L , Ayala F J
Published: July 2002

The hypothesis that morphological evolution may largely result from changes in gene regulation rather than gene structure has been difficult to test. Morphological differences among insects are often apparent in the cuticle structures produced. The dopa decarboxylase (Ddc) and alpha-methyldopa hypersensitive (amd) genes arose from an ancient gene duplication. In Drosophila, they have evolved nonoverlapping functions, including the production of distinct types of cuticle, and for Ddc, the production of the neurotransmitters, dopamine and serotonin. The amd gene is particularly active in the production of specialized flexible cuticles in the developing embryo. We have compared the pattern of amd expression in three Drosophila species. Several regions of expression conserved in all three species but, surprisingly, a unique domain of expression is found in Drosophila simulans that does occur in the closely related (2-5 million years) Drosophila melanogaster or in the more remote (40-50 million years) Drosophila virilis. The "sudden" appearance of a completely new and robust domain of expression provides a glimpse of evolutionary variation resulting from changes in regulation of structural gene expression.
Multicomponent diffusion in polymeric liquids.
Authors: Curtiss C F , Bird R B
Published: July 2002

It is shown how the phase-space kinetic theory of polymeric liquid mixtures leads to a set of extended Maxwell-Stefan equations describing multicomponent diffusion. This expression reduces to standard results for dilute solutions and for undiluted polymers. The polymer molecules are modeled as flexible bead-spring structures. To obtain the Maxwell-Stefan equations, the usual expression for the hydrodynamic drag force on a bead, used in previous kinetic theories, must be replaced by a new expression that accounts explicitly for bead-bead interactions between different molecules.
Pore-forming toxins trigger shedding of receptors for interleukin 6 and lipopolysaccharide.
Authors: Walev I , Vollmer P , Palmer M , Bhakdi S , Rose-John S
Published: July 2002

Cleavage of membrane-associated proteins with the release of biologically active macromolecules is an emerging theme in biology. However, little is known about the nature and regulation of the involved proteases or about the physiological inducers of the shedding process. We here report that rapid and massive shedding of the interleukin 6 receptor (IL-6R) and the lipopolysaccharide receptor (CD14) occurs from primary and transfected cells attacked by two prototypes of pore-forming bacterial toxins, streptolysin O and Escherichia coli hemolysin. Shedding is not induced by an streptolysin O toxin mutant which retains cell binding capacity but lacks pore-forming activity. The toxin-dependent cleavage site of the IL-6R was mapped to a position close to, but distinct from, that observed after stimulation with phorbol myristate acetate. Soluble IL-6R that was shed from toxin-treated cells bound its ligand and induced an IL-6-specific signal in cells that primarily lacked the IL-6R. Transsignaling by soluble IL-6R and soluble CD14 is known to dramatically broaden the spectrum of host cells for IL-6 and lipopolysaccharide, and is thus an important mechanism underlying their systemic inflammatory effects. Our findings uncover a novel mechanism that can help to explain the long-range detrimental action of pore-forming toxins in the host organism.
The sigma subunit of Escherichia coli RNA polymerase senses promoter spacing.
Authors: Dombroski A J , Johnson B D , Lonetto M , Gross C A
Published: July 2002

The promoters recognized by sigma 70, the primary sigma of Escherichia coli, consist of two highly conserved hexamers located at -10 and -35 bp from the start point of transcription, separated by a preferred spacing of 17 bp. sigma factors have two distinct DNA binding domains that recognize the two hexamer sequences. However, the component of RNA polymerase recognizing the length of the spacing between hexamers has not been determined. Using an equilibrium DNA binding competition assay, we demonstrate that a polypeptide of sigma 70 carrying both DNA binding domains is very sensitive to promoter spacing, whereas a sigma 70 polypeptide with only one DNA binding domain is not. Furthermore, a mutant sigma, selected for increasing transcription of the minimal lac promoter (18-bp spacer), has an altered response to promoter spacing in vivo and in vitro. Our data support the idea that sigma makes simultaneous, productive contacts at both the -10 and the -35 regions of the promoter and discerns the spacing between these conserved regions.
The weaver mutation of GIRK2 results in a loss of inwardly rectifying K+ current in cerebellar granule cells.
Authors: Surmeier D J , Mermelstein P G , Goldowitz D
Published: July 2002

The weaver mutation in mice results in a severe ataxia that is attributable to the degeneration of cerebellar granule cells and dopaminergic neurons in the substantia nigra. Recent genetic studies indicate that the GIRK2 gene is altered in weaver. This gene codes for a G-protein-activated, inwardly rectifying K+ channel protein (8). The mutation results in a single amino acid substitution (glycine-->serine) in the pore-forming H5 region of the channel. The functional consequences of this mutation appear to depend upon the co-expression of other GIRK subunits--leading to either a gain or loss of function. Here, we show that G-protein-activated inwardly rectifying K+ currents are significantly reduced in cerebellar granule cells from animals carrying the mutant allele. The reduction is most pronounced in homozygous neurons. These findings suggest that the death of neurons in weaver is attributable to the loss of GIRK2-mediated currents, not to the expression of a nonspecific cation current.
What is a linear process?
Authors: Bickel P J , Bühlmann P
Published: July 2002

We argue that given even an infinitely long data sequence, it is impossible (with any test statistic) to distinguish perfectly between linear and nonlinear processes (including slightly noisy chaotic processes). Our approach is to consider the set of moving-average (linear) processes and study its closure under a suitable metric. We give the precise characterization of this closure, which is unexpectedly large, containing nonergodic processes, which are Poisson sums of independent and identically distributed copies of a stationary process. Proofs of these results will appear elsewhere.
Glial cell line-derived neurotrophic factor promotes the development of adrenergic neurons in mouse neural crest cultures
Authors: Maxwell Gerald D. , Reid Kate , Elefanty Andrew , Bartlett Perry F. , Murphy Mark
Published: July 2002

Growth of mouse neural crest cultures in the presence of glial cell
line-derived neurotrophic factor (GDNF) resulted in a dramatic
dose-dependent increase in the number of tyrosine hydroxylase
(TH)-positive cells that developed when 5% chicken embryo extract was
present in the medium. In contrast, growth in the presence of bone
morphogenetic protein (BMP)-2, BMP-4, BMP-6, transforming growth factor
(TGF) β1, TGF-β2, and TGF-β3 elicited no increase in the number
of TH-positive cells. The TH-positive cells that developed in the
presence of GDNF had neuronal morphology and contained the middle and
low molecular weight neurofilament proteins. Numerous TH-negative cells
with the morphology of neurons also were observed in GDNF-treated
cultures. Analysis revealed that the period from 6 to 12 days
in
vitro
was the critical time for exposure to GDNF to generate
the increase in TH-positive cell number. The growth factors
neurotrophin-3 and fibroblast growth factor-2 elicited increases in the
number of TH-positive cells similar to that seen in response to GDNF.
In contrast, nerve growth factor was unable to substitute for GDNF.
These findings extend the previously reported biological activities of
GDNF by showing that it can act on mouse neural crest cultures to
promote the development of neurons.
Calexcitin: A signaling protein that binds calcium and GTP, inhibits potassium channels, and enhances membrane excitability
Authors: Nelson Thomas J. , Cavallaro Sebastiano , Yi Chu-Li , McPhie Donna , Schreurs Bernard G. , Gusev Pavel A. , Favit Antonella , Zohar Ofer , Kim Jeongho , Beushausen Sven , Ascoli Giorgio , Olds James , Neve Rachael , Alkon Daniel L.
Published: July 2002

A previously uncharacterized 22-kDa Ca
2+
-binding
protein that also binds guanosine nucleotides was characterized,
cloned, and analyzed by electrophysiological techniques. The cloned
protein, calexcitin, contains two EF-hands and also has homology with
GTP-binding proteins in the ADP ribosylation factor family. In addition
to binding two molecules of Ca
2+
, calexcitin bound GTP and
possessed GTPase activity. Calexcitin is also a high affinity substrate
for protein kinase C. Application of calexcitin to the inner surface of
inside-out patches of human fibroblast membranes, in the presence of
Ca
2+
and the absence of endogenous
Ca
2+
/calmodulin kinase type II or
protein kinase C activity, reduced the mean open time and mean open
probability of 115 ± 6 pS K
+
channels. Calexcitin
thus appears to directly regulate K
+
channels. When
microinjected into molluscan neurons or rabbit cerebellar Purkinje cell
dendrites, calexcitin was highly effective in enhancing membrane
excitability. Because calexcitin translocates to the cell membrane
after phosphorylation, calexcitin could serve as a
Ca
2+
-activated signaling molecule that increases cellular
excitability, which would in turn increase Ca
2+
influx through the membrane. This is also the first known instance of a
GTP-binding protein that binds Ca
2+
.
Targeted DNA recombination in vivo using an adenovirus carrying the cre recombinase gene.
Authors: Wang Y , Krushel L A , Edelman G M
Published: July 2002

Conditional gene expression and gene deletion are important experimental approaches for examining the functions of particular gene products in development and disease. The cre-loxP system from bacteriophage P1 has been used in transgenic animals to induce site-specific DNA recombination leading to gene activation or deletion. To regulate the recombination in a spatiotemporally controlled manner, we constructed a recombinant adenoviral vector, Adv/cre, that contained the cre recombinase gene under regulation of the herpes simplex virus thymidine kinase promoter. The efficacy and target specificity of this vector in mediating loxP-dependent recombination were analyzed in mice that had been genetically engineered to contain loxP sites in their genome. After intravenous injection of the Adv/cre vector into adult animals, the liver and spleen showed the highest infectivity of the adenovirus as well as the highest levels of recombination, whereas other tissues such as kidney, lung, and heart had lower levels of infection and recombination. Only trace levels of recombination were detected in the brain. However, when the Adv/cre vector was injected directly into specific regions of the adult brain, including the cerebral cortex, hippocampus, and cerebellum, recombination was detectable at the injection site. Furthermore, when the Adv/cre vector was injected into the forebrains of neonatal mice, the rearranged toxP locus from recombination could be detected in the injected regions for at least 8 weeks. Taken together, these results demonstrate that the Adv/cre vector expressing a functional cre protein is capable of mediating loxP-dependent recombination in various tissues and the recombined gene locus may in some cases be maintained for an extended period. The use of the adenovirus vector expressing cre combined with localized delivery to specific tissues may provide an efficient means to achieve conditional gene expression or knockout with precise spatiotemporal control.
STAT3 activation is a critical step in gp130-mediated terminal differentiation and growth arrest of a myeloid cell line.
Authors: Minami M , Inoue M , Wei S , Takeda K , Matsumoto M , Kishimoto T , Akira S
Published: July 2002

Myeloid leukemia M1 cells can be induced for growth arrest and terminal differentiation into macrophages in response to interleukin 6 (IL-6) or leukemia inhibitory factor (LIF). Recently, a large number of cytokines and growth factors have been shown to activate the Janus kinase (JAK)-signal transducer and activator of transcription (STAT) signaling pathway. In the case of IL-6 and LIF, which share a signal transducing receptor gp130, STAT3 is specifically tyrosine-phosphorylated and activated by stimulation with each cytokine in various cell types. To know the role of JAK-STAT pathway in M1 differentiation, we have constructed dominant negative forms of STAT3 and established M1 cell lines that constitutively express them. These M1 cells that overexpressed dominant negative forms showed no induction of differentiation-associated markers including Fc gamma receptors, ferritin light chain, and lysozyme after treatment with IL-6. Expression of either c-myb or c-myc was not downregulated. Furthermore, IL-6- and LIF-mediated growth arrest and apoptosis were completely blocked. Thus these findings demonstrate that STAT3 activation is the critical step in a cascade of events that leads to terminal differentiation of M1 cells.
RNA polymerase II transcription initiation: A structural view
Authors: Nikolov D. B. , Burley S. K.
Published: July 2002

In eukaryotes, RNA polymerase II transcribes messenger RNAs and
several small nuclear RNAs. Like RNA polymerases I and III, polymerase
II cannot act alone. Instead, general initiation factors
[transcription factor (TF) IIB, TFIID, TFIIE, TFIIF, and TFIIH]
assemble on promoter DNA with polymerase II, creating a large
multiprotein–DNA complex that supports accurate initiation. Another
group of accessory factors, transcriptional activators and
coactivators, regulate the rate of RNA synthesis from each gene in
response to various developmental and environmental signals. Our
current knowledge of this complex macromolecular machinery is reviewed
in detail, with particular emphasis on insights gained from structural
studies of transcription factors.
Clustering of meiotic double-strand breaks on yeast chromosome III
Authors: Baudat Frédéric , Nicolas Alain
Published: July 2002

In the yeast
Saccharomyces cerevisiae
, meiotic recombination is initiated by transient DNA double-strand breaks (DSBs) that are repaired by interaction of the broken chromosome with its homologue. To identify a large number of DSB sites and gain insight into the control of DSB formation at both the local and the whole chromosomal levels, we have determined at high resolution the distribution of meiotic DSBs along the 340 kb of chromosome III. We have found 76 DSB regions, mostly located in intergenic promoter-containing intervals. The frequency of DSBs varies at least 50-fold from one region to another. The global distribution of DSB regions along chromosome III is nonrandom, defining large (39–105 kb) chromosomal domains, both hot and cold. The distribution of these localized DSBs indicates that they are likely to initiate most crossovers along chromosome III, but some discrepancies remain to be explained.
CLARP, a death effector domain-containing protein interacts with caspase-8 and regulates apoptosis
Authors: Inohara Naohiro , Koseki Takeyoshi , Hu Yuanming , Chen Shu , Núñez Gabriel
Published: July 2002

We have identified and characterized CLARP, a
c
aspase-
l
ike
a
poptosis-
r
egulatory
p
rotein. Sequence analysis revealed that human CLARP contains two amino-terminal death effector domains fused to a carboxyl-terminal caspase-like domain. The structure and amino acid sequence of CLARP resemble those of caspase-8, caspase-10, and DCP2, a
Drosophila melanogaster
protein identified in this study. Unlike caspase-8, caspase-10, and DCP2, however, two important residues predicted to be involved in catalysis were lost in the caspase-like domain of CLARP. Analysis with fluorogenic substrates for caspase activity confirmed that CLARP is catalytically inactive. CLARP was found to interact with caspase-8 but not with FADD/MORT-1, an upstream death effector domain-containing protein of the Fas and tumor necrosis factor receptor 1 signaling pathway. Expression of CLARP induced apoptosis, which was blocked by the viral caspase inhibitor p35, dominant negative mutant caspase-8, and the synthetic caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-(OMe)-fluoromethylketone (zVAD-fmk). Moreover, CLARP augmented the killing ability of caspase-8 and FADD/MORT-1 in mammalian cells. The human
clarp
gene maps to 2q33. Thus, CLARP represents a regulator of the upstream caspase-8, which may play a role in apoptosis during tissue development and homeostasis.
Protein phosphatase 2C dephosphorylates and inactivates cystic fibrosis transmembrane conductance regulator
Authors: Travis Sue M. , Berger Herbert A. , Welsh Michael J.
Published: July 2002

cAMP-dependent phosphorylation activates the cystic fibrosis transmembrane conductance regulator (CFTR) in epithelia. However, the protein phosphatase (PP) that dephosphorylates and inactivates CFTR in airway and intestinal epithelia, two major sites of disease, is not certain. We found that in airway and colonic epithelia, neither okadaic acid nor FK506 prevented inactivation of CFTR when cAMP was removed. These results suggested that a phosphatase distinct from PP1, PP2A, and PP2B was responsible. Because PP2C is insensitive to these inhibitors, we tested the hypothesis that it regulates CFTR. We found that PP2Cα is expressed in airway and T84 intestinal epithelia. To test its activity on CFTR, we generated recombinant human PP2Cα and found that it dephosphorylated CFTR and an R domain peptide
in vitro
. Moreover, in cell-free patches of membrane, addition of PP2Cα inactivated CFTR Cl
−
channels; reactivation required readdition of kinase. Finally, coexpression of PP2Cα with CFTR in epithelia reduced the Cl
−
current and increased the rate of channel inactivation. These results suggest that PP2C may be the okadaic acid-insensitive phosphatase that regulates CFTR in human airway and T84 colonic epithelia. It has been suggested that phosphatase inhibitors could be of therapeutic value in cystic fibrosis; our data suggest that PP2C may be an important phosphatase to target.
Direct measurement of nitric oxide generation from nitric oxide synthase
Authors: Xia Yong , Zweier Jay L.
Published: July 2002

Although nitric oxide synthase (NOS) is widely considered as the major source of NO in biological cells and tissues, direct evidence demonstrating NO formation from the purified enzyme has been lacking. It was recently reported that NOS does not synthesize NO, but rather generates nitroxyl anion (NO
−
) that is subsequently converted to NO by superoxide dismutase (SOD). To determine if NOS synthesizes NO, electron paramagnetic resonance (EPR) spectroscopy was applied to directly measure NO formation from purified neuronal NOS. In the presence of the NO trap Fe
2+
-
N
-methyl-
d
-glucamine dithiocarbamate, NO gives rise to characteristic EPR signals with
g
= 2.04 and
a
N
= 12.7 G, whereas NO
−
is undetectable. In the presence of
l
-arginine (
l
-Arg) and cofactors, NOS generated prominent NO signals. This NO generation did not require SOD, and it was blocked by the specific NOS inhibitor
N
-nitro-
l
-arginine methyl ester. Isotope-labeling experiments with
l
-[
15
N]Arg further demonstrated that NOS-catalyzed NO arose from the guanidino nitrogen of
l
-Arg. Measurement of the time course of NO formation demonstrated that it paralleled that of
l
-citrulline. The conditions used in the prior study were shown to result in potent superoxide generation, and this may explain the failure to measure NO formation in the absence of SOD. These experiments provide unequivocal evidence that NOS does directly synthesize NO from
l
-Arg.
Global variation in the genetic and biochemical basis of diamondback moth resistance to
Bacillus thuringiensis
Authors: Tabashnik Bruce E. , Liu Yong-Biao , Malvar Thomas , Heckel David G. , Masson Luke , Ballester Victoria , Granero Francisco , Ménsua José L. , Ferré Juan
Published: July 2002

Insecticidal proteins from the soil bacterium
Bacillus thuringiensis
(Bt) are becoming a cornerstone of ecologically sound pest management. However, if pests quickly adapt, the benefits of environmentally benign Bt toxins in sprays and genetically engineered crops will be short-lived. The diamondback moth (
Plutella xylostella
) is the first insect to evolve resistance to Bt in open-field populations. Here we report that populations from Hawaii and Pennsylvania share a genetic locus at which a recessive mutation associated with reduced toxin binding confers extremely high resistance to four Bt toxins. In contrast, resistance in a population from the Philippines shows multilocus control, a narrower spectrum, and for some Bt toxins, inheritance that is not recessive and not associated with reduced binding. The observed variation in the genetic and biochemical basis of resistance to Bt, which is unlike patterns documented for some synthetic insecticides, profoundly affects the choice of strategies for combating resistance.
Localization and activity of lysyl oxidase within nuclei of fibrogenic cells
Authors: Li Wande , Nellaiappan Kaliappanadar , Strassmaier Timothy , Graham Lila , Thomas Kathleen M. , Kagan Herbert M.
Published: July 2002

Lysyl oxidase (EC
1.4.3.13
) oxidizes peptidyl lysine to peptidyl aldehyde residues within collagen and elastin, thus initiating formation of the covalent cross-linkages that insolubilize these extracellular proteins. Recent findings raise the possibility that this enzyme may also function intracellularly. The present study provides evidence by immunocytochemical confocal microscopy, Western blot analysis, enzyme assays, and chemical analyses for lysyl oxidase reaction products that this enzyme is present and active within rat vascular smooth muscle cell nuclei. Confocal microscopy indicates its presence within nuclei of 3T3 fibroblasts, as well.
Crystal structure of desheptapeptide(B24–B30)insulin at 1.6 Å resolution: Implications for receptor binding
Authors: Bao Su-jin , Xie Dian-lin , Zhang Ji-ping , Chang Wen-rui , Liang Dong-cai
Published: July 2002

The crystal structure of desheptapeptide (B24–B30) insulin (DHPI), a virtually inactive analog of insulin, was determined at 1.6 Å resolution. In the refined structure model, DHPI retains three α-helices (A1–A8, A12–A18, and B9–B19) as its structural framework, while great conformational changes occur in the N and C termini of B-chain. The β-turn, which lies in B20–B30 in insulin and insulin analogs with high potency, no longer exists in DHPI. Relative motion is observed among the three α-helices, each as a rigid functional group. In contrast, a region covering B5–B6 and A6–A11 exhibits a relatively stable conformation. We interpret our results as identifying: (
i
) the importance of β-turn in determining the receptor-binding potency of insulin and (
ii
) a leading role of Phe
B24
in maintaining the β-turn structure.
Chlorophyll
a
dimer: A possible primary electron donor for the photosystem II
Authors: Boussaad S. , Tazi A. , Leblanc R. M.
Published: July 2002

In the photosynthetic membrane, there is a particular aggregated state for the chlorophyll
a
(Chl
a
) molecules with a specific arrangement responsible for the high efficiency of energy conversion. Chl
a
monolayers, transferred onto solid substrates, are systems that potentially can mimic the packing of the
in vivo
system. The association of Chl
a
in the monolayer results in the formation of dimers with an average size of 3.00 ± 0.15 nm. Considering the organization of the dimers, we assume that P680 is a dimer with the (anti) parallel transition moments of the constituent. The Chl
a
macrocycles most likely are tilted to each other by 30° with respect to the membrane plane.
MgATP activates the β cell K
ATP
channel by interaction with its SUR1 subunit
Authors: Gribble Fiona M. , Tucker Stephen J. , Haug Trude , Ashcroft Frances M.
Published: July 2002

ATP-sensitive potassium (K
ATP
) channels in the pancreatic β cell membrane mediate insulin release in response to elevation of plasma glucose levels. They are open at rest but close in response to glucose metabolism, producing a depolarization that stimulates Ca
2+
influx and exocytosis. Metabolic regulation of K
ATP
channel activity currently is believed to be mediated by changes in the intracellular concentrations of ATP and MgADP, which inhibit and activate the channel, respectively. The β cell K
ATP
channel is a complex of four Kir6.2 pore-forming subunits and four SUR1 regulatory subunits: Kir6.2 mediates channel inhibition by ATP, whereas the potentiatory action of MgADP involves the nucleotide-binding domains (NBDs) of SUR1. We show here that MgATP (like MgADP) is able to stimulate K
ATP
channel activity, but that this effect normally is masked by the potent inhibitory effect of the nucleotide. Mg
2+
caused an apparent reduction in the inhibitory action of ATP on wild-type K
ATP
channels, and MgATP actually activated K
ATP
channels containing a mutation in the Kir6.2 subunit that impairs nucleotide inhibition (R50G). Both of these effects were abolished when mutations were made in the NBDs of SUR1 that are predicted to abolish MgATP binding and/or hydrolysis (D853N, D1505N, K719A, or K1384M). These results suggest that, like MgADP, MgATP stimulates K
ATP
channel activity by interaction with the NBDs of SUR1. Further support for this idea is that the ATP sensitivity of a truncated form of Kir6.2, which shows functional expression in the absence of SUR1, is unaffected by Mg
2+
.
A resistant genetic background leading to incomplete penetrance of intestinal neoplasia and reduced loss of heterozygosity in

Apc
Min

/+ mice
Authors: Shoemaker Alex R. , Moser Amy R. , Midgley Carol A. , Clipson Linda , Newton Michael A. , Dove William F.
Published: July 2002

Previous studies of
Min
/+ (multiple intestinal neoplasia) mice on a sensitive genetic background, C57BL/6 (B6), showed that adenomas have lost heterozygosity for the germ-line

Apc
Min

mutation in the
Apc
(adenomatous polyposis coli) gene. We now report that on a strongly resistant genetic background, AKR/J (AKR),
Min
-induced adenoma multiplicity is reduced by about two orders of magnitude compared with that observed on the B6 background. Somatic treatment with a strong mutagen increases tumor number in AKR
Min
/+ mice in an age-dependent manner, similar to results previously reported for B6
Min
/+ mice. Immunohistochemical analyses indicate that Apc expression is suppressed in all intestinal tumors from both untreated and treated AKR
Min
/+ mice. However, the mechanism of
Apc
inactivation in AKR
Min
/+ mice often differs from that observed for B6
Min
/+ mice. Although loss of heterozygosity is observed in some tumors, a significant percentage of tumors showed neither loss of heterozygosity nor
Apc
truncation mutations. These results extend our understanding of the effects of genetic background on
Min
-induced tumorigenesis in several ways. First, the AKR strain carries modifiers of
Min
in addition to
Mom1
. This combination of AKR modifiers can almost completely suppress spontaneous intestinal tumorigenesis associated with the
Min
mutation. Second, even on such a highly resistant genetic background, tumor formation continues to involve an absence of Apc function. The means by which Apc function is inactivated is affected by genetic background. Possible scenarios are discussed.
Cloning and localization of two multigene receptor families in goldfish olfactory epithelium
Authors: Cao Yanxiang , Oh Bryan C. , Stryer Lubert
Published: July 2002

Goldfish reproduction is coordinated by pheromones that are released by ovulating females and detected by males. Two highly potent pheromones, a dihydroxyprogesterone and a prostaglandin, previously have been identified, and their effects on goldfish behavior have been studied in depth. We have cloned goldfish olfactory epithelium cDNAs belonging to two multigene G-protein coupled receptor families as a step toward elucidating the molecular basis of pheromone recognition. One gene family (GFA) consists of homologs of putative odorant receptors (≈320 residues) found in the olfactory epithelium of other fish and mammals. The other family (GFB) consists of homologs of putative pheromone receptors found in the vomeronasal organ (VNO) of mammals and also in the nose of pufferfish. GFB receptors (≈840 residues) are akin to the V2R family of VNO receptors, which possess a large extracellular N-terminal domain and are homologs of calcium-sensing and metabotropic glutamate receptors.
In situ
hybridization showed that the two families of goldfish receptors are differentially expressed in the olfactory epithelium. GFB mRNA is abundant in rather compact cells whose nuclei are near the apical surface. In contrast, GFA mRNA is found in elongated cells whose nuclei are positioned deeper in the epithelium. Our findings support the hypothesis that the separate olfactory organ and VNO of terrestrial vertebrates arose in evolution by the segregation of distinct classes of neurons that were differentially positioned in the olfactory epithelium of a precursor aquatic vertebrate.
CM101-mediated recovery of walking ability in adult mice paralyzed by spinal cord injury
Authors: Wamil Artur W. , Wamil Barbara D. , Hellerqvist Carl G.
Published: July 2002

CM101, an antiangiogenic polysaccharide derived from group B streptococcus, was administered by i.v. injection 1 hr post-spinal-cord crush injury in an effort to prevent inflammatory angiogenesis and gliosis (scarring) in a mouse model. We postulated that gliosis would sterically prevent the reestablishment of neuronal connectivity; thus, treatment with CM101 was repeated every other day for five more infusions for the purpose of facilitating regeneration of neuronal function. Twenty-five of 26 mice treated with CM101 survived 28 days after surgery, and 24 of 26 recovered walking ability within 2–12 days. Only 6 of 14 mice in the control groups survived 24 hr after spinal cord injury, and none recovered function in paralyzed limbs. MRI analysis of injured untreated and treated animals showed that CM101 reduced the area of damage at the site of spinal cord compression, which was corroborated by histological analysis of spinal cord sections from treated and control animals. Electrophysiologic measurements on isolated central nervous system and neurons in culture showed that CM101 protected axons from Wallerian degeneration; reversed γ-aminobutyrate-mediated depolarization occurring in traumatized neurons; and improved recovery of neuronal conductivity of isolated central nervous system in culture.
Mitochondrial stress protein recognition of inactivated dehydrogenases during mammalian cell death
Authors: Bruschi Sam A. , Lindsay J. Gordon , Crabb John W.
Published: July 2002

The mammalian renal toxicant tetrafluoroethylcysteine (TFEC) is metabolized to a reactive intermediate that covalently modifies the lysine residues of a select group of mitochondrial proteins, forming difluorothioamidyl lysine protein adducts. Cellular damage is initiated by this process and cell death ensues. NH
2
-terminal sequence analysis of purified mitochondrial proteins containing difluorothioamidyl lysine adducts identified the lipoamide succinyltransferase and dihydrolipoamide dehydrogenase subunits of the α-ketoglutarate dehydrogenase complex (αKGDH), a key regulatory component of oxidative metabolism, as targets for TFEC action. Adduct formation resulted in marked inhibition of αKGDH enzymatic activity, whereas the related pyruvate dehydrogenase complex was unmodified by TFEC and its activity was not inhibited
in vivo
. Covalent modification of αKGDH subunits also resulted in interactions with mitochondrial chaperonin HSP60
in vivo
and with HSP60 and mitochondrial HSP70
in vitro
. These observations confirm the role of mammalian stress proteins in the recognition of abnormal proteins and provide supporting evidence for reactive metabolite-induced cell death by modification of critical protein targets.