bluuebunny/crossref_metadata_2025 huggingface dataset:
Chronic intracellular infection of alfalfa nodules by
Sinorhizobium meliloti
requires correct lipopolysaccharide core
Authors: Campbell Gordon R. O. , Reuhs Bradley L. , Walker Graham C.
Published: July 2002

Our analyses of lipopolysaccharide mutants of
Sinorhizobium meliloti
offer insights into how this bacterium establishes the chronic intracellular infection of plant cells that is necessary for its nitrogen-fixing symbiosis with alfalfa. Derivatives of
S. meliloti
strain Rm1021 carrying an
lpsB
mutation are capable of colonizing curled root hairs and forming infection threads in alfalfa in a manner similar to a wild-type strain. However, developmental abnormalities occur in the bacterium and the plant at the stage when the bacteria invade the plant nodule cells. Loss-of-function
lpsB
mutations, which eliminate a protein of the glycosyltransferase I family, cause striking changes in the carbohydrate core of the lipopolysaccharide, including the absence of uronic acids and a 40-fold relative increase in xylose. We also found that
lpsB
mutants were sensitive to the cationic peptides melittin, polymyxin B, and poly-
l
-lysine, in a manner that paralleled that of
Brucella
abortus
lipopolysaccharide mutants. Sensitivity to components of the plant's innate immune system may be part of the reason that this mutant is unable to properly sustain a chronic infection within the cells of its host-plant alfalfa.
Profound defects in pancreatic β-cell function in mice with combined heterozygous mutations in
Pdx-1
,
Hnf-1
α, and
Hnf-3
β
Authors: Shih David Q. , Heimesaat Markus , Kuwajima Satoru , Stein Roland , Wright Christopher V. E. , Stoffel Markus
Published: July 2002

Defects in pancreatic β-cell function contribute to the development of type 2 diabetes, a polygenic disease that is characterized by insulin resistance and compromised insulin secretion. Hepatocyte nuclear factors (HNFs) -1α, -3β, -4α, and Pdx-1 contribute in the complex transcriptional circuits within the pancreas that are involved in β-cell development and function. In mice, a heterozygous mutation in
Pdx-1
alone, but not
Hnf-1
α
+/−
,
Hnf-3
β
+/−
, or
Hnf-4
α
+/−
, causes impaired glucose-stimulated insulin secretion in mice. To investigate the possible functional relationships between these transcription factors on β-cell activity
in vivo
, we generated mice with the following combined heterozygous mutations:
Pdx-1
+/−
/
Hnf-1α
+/−
,
Pdx-1
+/−
/
Hnf-3β
+/−
,
Pdx-1
+/−
/
Hnf-4α
+/−
,
Hnf-1α
+/−
/
Hnf-4α
+/−
, and
Hnf-3β
+/−
/
Hnf-4α
+/−
. The greatest loss in function was in combined heterozygous null alleles of
Pdx-1
and
Hnf-1α
(
Pdx-1
+/−
/
Hnf-1α
+/−
), or
Pdx-1
and
Hnf-3β
(
Pdx-1
+/−
/
Hnf-3β
+/−
). Both double mutants develop progressively impaired glucose tolerance and acquire a compromised first- and second-phase insulin secretion profile in response to glucose compared with
Pdx-1
+/−
mice alone. The loss in β-cell function in
Pdx-1
+/−
/
Hnf-3β
+/−
mice was associated with decreased expression of
Nkx-6.1,
glucokinase (Gck), aldolase B (
aldo-B
), and insulin, whereas
Nkx2.2, Nkx-6.1, Glut-2, Gck, aldo-B,
the liver isoform of pyruvate kinase, and insulin expression was reduced in
Pdx-1
+/−
/
Hnf-1α
+/−
mice. The islet cell architecture was also abnormal in
Pdx-1
+/−
/
Hnf-3β
+/−
and
Pdx-1
+/−
/
Hnf-1α
+/−
mice, with glucagon-expressing cells scattered throughout the islet, a defect that may be connected to decreased E-cadherin expression. Our data suggest that functional interactions between key islet regulatory factors play an important role in maintaining islet architecture and β-cell function. These studies also established polygenic mouse models for investigating the mechanisms contributing to β-cell dysfunction in diabetes.
Overexpression of the LAR (leukocyte antigen-related) protein-tyrosine phosphatase in muscle causes insulin resistance
Authors: Zabolotny Janice M. , Kim Young-Bum , Peroni Odile D. , Kim Jason K. , Pani Michael A. , Boss Olivier , Klaman Lori D. , Kamatkar Shubhangi , Shulman Gerald I. , Kahn Barbara B. , Neel Benjamin G.
Published: July 2002

Previous reports indicate that the expression and/or activity of
the protein-tyrosine phosphatase (PTP) LAR are increased in
insulin-responsive tissues of obese, insulin-resistant humans and
rodents, but it is not known whether these alterations contribute to
the pathogenesis of insulin resistance. To address this question, we
generated transgenic mice that overexpress human LAR, specifically in
muscle, to levels comparable to those reported in insulin-resistant
humans. In LAR-transgenic mice, fasting plasma insulin was increased
2.5-fold compared with wild-type controls, whereas fasting glucose was
normal. Whole-body glucose disposal and glucose uptake into muscle
in vivo
were reduced by 39–50%. Insulin injection
resulted in normal tyrosyl phosphorylation of the insulin receptor and
insulin receptor substrate 1 (IRS-1) in muscle of transgenic mice.
However, phosphorylation of IRS-2 was reduced by 62%, PI3′ kinase
activity associated with phosphotyrosine, IRS-1, or IRS-2 was reduced
by 34–57%, and association of p85α with both IRS proteins was
reduced by 39–52%. Thus, overexpression of LAR in muscle causes
whole-body insulin resistance, most likely due to dephosphorylation of
specific regulatory phosphotyrosines on IRS proteins. Our data suggest
that increased expression and/or activity of LAR or related PTPs in
insulin target tissues of obese humans may contribute to the
pathogenesis of insulin resistance.
Ccm1
, a regulatory gene controlling the induction of a carbon-concentrating mechanism in
Chlamydomonas reinhardtii
by sensing CO
2
availability
Authors: Fukuzawa Hideya , Miura Kenji , Ishizaki Kimitsune , Kucho Ken-ichi , Saito Tatsuaki , Kohinata Tsutomu , Ohyama Kanji
Published: July 2002

Aquatic photosynthetic organisms, including the green alga
Chlamydomonas reinhardtii
, induce a set of genes for a
carbon-concentrating mechanism (CCM) to acclimate to
CO
2
-limiting conditions. This acclimation is modulated by
some mechanisms in the cell to sense CO
2
availability.
Previously, a high-CO
2
-requiring mutant C16 defective in an
induction of the CCM was isolated from
C. reinhardtii
by
gene tagging. By using this pleiotropic mutant, we isolated a nuclear
regulatory gene,
Ccm1
, encoding a 699-aa hydrophilic
protein with a putative zinc-finger motif in its N-terminal region and
a Gln repeat characteristic of transcriptional activators. Introduction
of
Ccm1
into this mutant restored an active carbon
transport through the CCM, development of a pyrenoid structure in the
chloroplast, and induction of a set of CCM-related genes. That a
5,128-base
Ccm1
transcript and also the translation
product of 76 kDa were detected in both high- and low-CO
2
conditions suggests that CCM1 might be modified posttranslationally.
These data indicate that
Ccm1
is essential to control
the induction of CCM by sensing CO
2
availability in
Chlamydomonas
cells. In addition, complementation assay
and identification of the mutation site of another pleiotropic mutant,
cia5
, revealed that His-54 within the putative
zinc-finger motif of the CCM1 is crucial to its regulatory function.
Xpbx1b
and
Xmeis1b
play a collaborative role in hindbrain and neural crest gene expression in
Xenopus
embryos
Authors: Maeda Ryu , Ishimura Akihiko , Mood Kathleen , Park Eui Kyun , Buchberg Arthur M. , Daar Ira O.
Published: July 2002

Pbx1 is a homeodomain protein that functions in complexes with other homeodomain-containing proteins to regulate gene expression during embryogenesis and oncogenesis. Pbx proteins bind DNA cooperatively as heterodimers or higher order complexes with Meis family members and Hox proteins and are believed to specify cell identity during development. Here, we present evidence that Pbx1, in partnership with Meis1b, can regulate posterior neural markers and neural crest marker genes during
Xenopus
development. A
Xenopus
homolog of the Pbx1b homeodomain protein was isolated and shown to be expressed throughout embryogenesis.
Xpbx1b
expression overlaps with
Xmeis1
in several areas, including the lateral neural folds, caudal branchial arch, hindbrain, and optic cup. When ectopically expressed, Xpbx1b can synergize with Xmeis1b to promote posterior neural and neural crest gene expression in ectodermal explants. Further, a physical interaction between these two homeodomain proteins is necessary for induction of these genes in embryonic tissue. In addition, coexpression of Xmeis1b and Xpbx1b leads to a prominent shift in the localization of Xmeis1b from the cytoplasm to the nucleus, suggesting that nuclear transport or retention of Xmeis1b may depend upon Xpbx1b. Finally, expression of a mutant construct in which Xpbx1b protein is fused to the repressor domain from
Drosophila
Engrailed inhibits posterior neural and neural crest gene expression. These data indicate that Xpbx1b and its partner, Xmeis1b, function in a transcriptional activation complex during hindbrain and neural crest development.
NF-κB inhibition causes spontaneous apoptosis in Epstein–Barr virus-transformed lymphoblastoid cells
Authors: Cahir-McFarland Ellen D. , Davidson David M. , Schauer Stephanie L. , Duong Jimmy , Kieff Elliott
Published: July 2002

Epstein–Barr virus (EBV) transforms B lymphocytes into lymphoblastoid cell lines usurping the Notch and tumor necrosis factor receptor pathways to effect transcription including NF-κB activation. To determine whether NF-κB activity is essential in the growth and survival of EBV-transformed lymphoblastoid cell lines, a nondegradable IκBα mutant was expressed under tetracycline regulation. Despite continued Bcl-2 and Bcl-x/L expression, NF-κB inhibition induced apoptosis as evidenced by poly(ADP-ribose) polymerase cleavage, nuclear condensation and fragmentation, and hypodiploid DNA content. Both caspase 3 and 8 activation and loss of mitochondrial membrane potential were observed in apoptotic cells. However, caspase inhibition failed to block apoptosis. These experiments indicate that NF-κB inhibitors may be useful in the therapy of EBV-induced cellular proliferation.
Assembling the glycopeptide antibiotic scaffold: The biosynthesis of from
Streptomyces toyocaensis
NRRL15009
Authors: Pootoolal Jeff , Thomas Michael G. , Marshall C. Gary , Neu John M. , Hubbard Brian K. , Walsh Christopher T. , Wright Gerard D.
Published: July 2002

The glycopeptide antibiotics vancomycin and teicoplanin are vital components of modern anti-infective chemotherapy exhibiting outstanding activity against Gram-positive pathogens including members of the genera
Streptococcus
,
Staphylococcus
, and
Enterococcus
. These antibiotics also provide fascinating examples of the chemical and associated biosynthetic complexity exploitable in the synthesis of natural products by actinomycetes group of bacteria. We report the sequencing and annotation of the biosynthetic gene cluster for the glycopeptide antibiotic
A47934
from
Streptomyces toyocaensis
NRRL15009, the first complete sequence for a teicoplanin class glycopeptide. The cluster includes 34 ORFs encompassing 68 kb and includes all of the genes predicted to be required to synthesize
A47934
and regulate its biosynthesis. The gene cluster also contains ORFs encoding enzymes responsible for glycopeptide resistance. This role was confirmed by insertional inactivation of the
d
-Ala-
d
-lactate ligase,
vanAst
, which resulted in the predicted
A47934
-sensitive phenotype and impaired antibiotic biosynthesis. These results provide increased understanding of the biosynthesis of these complex natural products.
Direct DNA binding by Brca1
Authors: Paull Tanya T. , Cortez David , Bowers Blair , Elledge Stephen J. , Gellert Martin
Published: July 2002

The tumor suppressor Brca1 plays an important role in protecting
mammalian cells against genomic instability, but little is known about
its modes of action. In this work we demonstrate that recombinant human
Brca1 protein binds strongly to DNA, an activity conferred by a domain
in the center of the Brca1 polypeptide. As a result of this binding,
Brca1 inhibits the nucleolytic activities of the Mre11/Rad50/Nbs1
complex, an enzyme implicated in numerous aspects of double-strand
break repair. Brca1 displays a preference for branched DNA structures
and forms protein–DNA complexes cooperatively between multiple DNA
strands, but without DNA sequence specificity. This fundamental
property of Brca1 may be an important part of its role in DNA repair
and transcription.
Recursive partitioning for tumor classification with gene expression microarray data
Authors: Zhang Heping , Yu Chang-Yung , Singer Burton , Xiong Momiao
Published: July 2002

Precise classification of tumors is critically important for cancer
diagnosis and treatment. It is also a scientifically challenging task.
Recently, efforts have been made to use gene expression profiles to
improve the precision of classification, with limited success. Using a
published data set for purposes of comparison, we introduce a
methodology based on classification trees and demonstrate that it is
significantly more accurate for discriminating among distinct colon
cancer tissues than other statistical approaches used heretofore. In
addition, competing classification trees are displayed, which suggest
that different genes may coregulate colon cancers.
Recombinant tobacco mosaic virus movement protein is an RNA-binding, α-helical membrane protein
Authors: Brill Laurence M. , Nunn Rashmi S. , Kahn Theodore W. , Yeager Mark , Beachy Roger N.
Published: July 2002

The 30-kDa movement protein (MP) is essential for cell–cell spread of tobacco mosaic virus
in planta
. To explore the structural properties of MP, the full-length recombinant MP gene was expressed in
Escherichia coli
, and one-step purification from solubilized inclusion bodies was accomplished by using anion exchange chromatography. Soluble MP was maintained at >4 mg/ml without aggregation and displayed ≈70% α-helical conformation in the presence of urea and SDS. A trypsin-resistant core domain of the MP had tightly folded tertiary structure, whereas 18 aa at the C terminus of the monomer were rapidly removed by trypsin. Two hydrophobic regions within the core were highly resistant to proteolysis. Based on results of CD spectroscopy, trypsin treatment, and MS, we propose a topological model in which MP has two putative α-helical transmembrane domains and a protease-sensitive carboxyl terminus.
Activation of polyamine catabolism in transgenic rats induces acute pancreatitis
Authors: Alhonen Leena , Parkkinen Jyrki J. , Keinänen Tuomo , Sinervirta Riitta , Herzig Karl-Heinz , Jänne Juhani
Published: July 2002

Polyamines are required for optimal growth and function of cells. Regulation of their cellular homeostasis is therefore tightly controlled. The key regulatory enzyme for polyamine catabolism is the spermidine/spermine
N
1
-acetyltransferase (SSAT). Depletion of cellular polyamines has been associated with inhibition of growth and programmed cell death. To investigate the physiological function SSAT, we generated a transgenic rat line overexpressing the SSAT gene under the control of the inducible mouse metallothionein I promoter. Administration of zinc resulted in a marked induction of pancreatic SSAT, overaccumulation of putrescine, and appearance of
N
1
-acetylspermidine with extensive depletion of spermidine and spermine in transgenic animals. The activation of pancreatic polyamine catabolism resulted in acute pancreatitis. In nontransgenic animals, an equal dose of zinc did not affect pancreatic polyamine pools, nor did it induce pancreatitis. Acetylated polyamines, products of the SSAT-catalyzed reaction, are metabolized further by the polyamine oxidase (PAO) generating hydrogen peroxide, which might cause or contribute to the pancreatic inflammatory process. Administration of specific PAO inhibitor, MDL72527 [
N
1
,
N
2
-bis(2,3-butadienyl)-1,4-butanediamine], however, did not affect the histological score of the pancreatitis. Induction of SSAT by the polyamine analogue
N
1
,
N
11
-diethylnorspermine reduced pancreatic polyamines levels only moderately and without signs of organ inflammation. In contrast, the combination of
N
1
,
N
11
-diethylnorspermine with MDL72527 dramatically activated SSAT, causing profound depletion of pancreatic polyamines and acute pancreatitis. These results demonstrate that acute induction of SSAT leads to pancreatic inflammation, suggesting that sufficient pools of higher polyamine levels are essential to maintain pancreatic integrity. This inflammatory process is independent of the production of hydrogen peroxide by PAO.
Directed evolution of a (βα)
8
-barrel enzyme to catalyze related reactions in two different metabolic pathways
Authors: Jürgens Catharina , Strom Alexander , Wegener Dennis , Hettwer Stefan , Wilmanns Matthias , Sterner Reinhard
Published: July 2002

Enzymes participating in different metabolic pathways often have similar catalytic mechanisms and structures, suggesting their evolution from a common ancestral precursor enzyme. We sought to create a precursor-like enzyme for
N
′-[(5′-phosphoribosyl)formimino]-5-aminoimidazole-4-carboxamide ribonucleotide (ProFAR) isomerase (HisA; EC
5.3.1.16
) and phosphoribosylanthranilate (PRA) isomerase (TrpF; EC
5.3.1.24
), which catalyze similar reactions in the biosynthesis of the amino acids histidine and tryptophan and have a similar (βα)
8
-barrel structure. Using random mutagenesis and selection, we generated several HisA variants that catalyze the TrpF reaction both
in vivo
and
in vitro
, and one of these variants retained significant HisA activity. A more detailed analysis revealed that a single amino acid exchange could establish TrpF activity on the HisA scaffold. These findings suggest that HisA and TrpF may have evolved from an ancestral enzyme of broader substrate specificity and underscore that (βα)
8
-barrel enzymes are very suitable for the design of new catalytic activities.
The U
S
3 protein kinase of herpes simplex virus 1 mediates the posttranslational modification of BAD and prevents BAD-induced programmed cell death in the absence of other viral proteins
Authors: Munger Joshua , Roizman Bernard
Published: July 2002

Earlier studies have shown that the d120 mutant of herpes simplex virus 1, which lacks both copies of the α4 gene, induces apoptosis in all cell lines tested. In some cell lines d120-induced apoptosis, manifested by the release of cytochrome
c
, activation of caspase 3, and fragmentation of cellular DNA, is blocked by the overexpression of Bcl-2. In these cells viral protein kinase U
S
3 delivered in
trans
blocks apoptosis induced by the mutant virus at a premitochondrial stage. We report that the U
S
3 protein kinase targets the pro-apoptotic BAD member of the Bcl-2 family. Specifically, the U
S
3 protein kinase mediates a posttranslational modification of BAD and blocks its cleavage, which is reported to activate apoptosis. Thus, U
S
3 protein kinase is the sole viral protein required to block activation of caspase 3, prevent cleavage of poly(ADP-ribose) polymerase, and block fragmentation of cellular DNA induced by BAD.
Nucleocytoplasmic translocation of Stat1 is regulated by a leucine-rich export signal in the coiled-coil domain
Authors: Begitt Andreas , Meyer Thomas , van Rossum Marleen , Vinkemeier Uwe
Published: July 2002

Signal transducer and activator of transcription (Stat) proteins are latent transcription factors that reside in the cytoplasm before activation. On cytokine-induced tyrosine phosphorylation, these molecules dimerize and accumulate transiently in the nucleus. No specific signals mediating these processes have been identified to date. In this report, we examine the nuclear export of Stat1. We find that treatment of cells with the export inhibitor leptomycin B does not affect steady-state localization of Stat1 but impedes nuclear export after IFNγ-induced nuclear accumulation. We identify a conserved leucine-rich helical segment in the coiled-coil domain of Stat1, which is responsible for the efficient nuclear export of this protein. Mutation of two hallmark leucines within this segment greatly attenuate the back transport of Stat1 in the cytoplasm. When fused to a carrier protein, the Stat1 export sequence can mediate nuclear export after intranuclear microinjection. We show that prolonging the nuclear presence of Stat1 by inhibiting nuclear export reduces the transcriptional response to stimulation with IFNγ. These data suggest that Stats are actively exported from the nucleus via several separate pathways and link this activity to transcriptional activation.
Neural roles for heme oxygenase: Contrasts to nitric oxide synthase
Authors: Barañano David E. , Snyder Solomon H.
Published: July 2002

The heme oxygenase (HO) and nitric oxide (NO) synthase (NOS)
systems display notable similarities as well as differences. HO and NOS
are both oxidative enzymes using NADPH as an electron donor. The
constitutive forms of the enzyme are differentially activated, with
calcium entry stimulating NOS by binding to calmodulin,
whereas calcium entry activates protein kinase C to phosphorylate and
activate HO2. Although both NO and carbon monoxide (CO) stimulate
soluble guanylyl cyclase to form cGMP, NO also S-nitrosylates selected
protein targets. Both involve constitutive and inducible biosynthetic
enzymes. However, functions of the inducible forms are virtual
opposites. Macrophage-inducible NOS generates NO to kill other cells,
whereas HO1 generates bilirubin to exert antioxidant cytoprotective
effects and also provides cytoprotection by facilitating iron extrusion
from cells. The neuronal form of HO, HO2, is also cytoprotective.
Normally, neural NO in the brain seems to exert some sort of behavioral
inhibition. However, excess release of NO in response to glutamate's
N
-methyl-
d
-aspartate receptor activation
leads to stroke damage. On the other hand, massive neuronal firing
during a stroke presumably activates HO2, leading to neuroprotective
actions of bilirubin. Loss of this neuroprotection after HO inhibition
by mutant forms of amyloid precursor protein may mediate neurotoxicity
in Familial Alzheimer's Disease. NO and CO both appear to be
neurotransmitters in the brain and peripheral autonomic nervous system.
They also are physiologic endothelial-derived relaxing factors for
blood vessels. In the gastrointestinal pathway, NO and CO appear to
function as coneurotransmitters, both stimulating soluble guanylyl
cyclase to cause smooth muscle relaxation.
Sensory experience and sensory activity regulate chemosensory receptor gene expression in
Caenorhabditis elegans
Authors: Peckol Erin L. , Troemel Emily R. , Bargmann Cornelia I.
Published: July 2002

Changes in the environment cause both short-term and long-term
changes in an animal's behavior. Here we show that specific sensory
experiences cause changes in chemosensory receptor gene expression that
may alter sensory perception in the nematode
Caenorhabditis elegans
. Three predicted
chemosensory receptor genes expressed in the ASI chemosensory neurons,
srd-1
,
str-2
, and
str-3
,
are repressed by exposure to the dauer pheromone, a signal of crowding.
Repression occurs at pheromone concentrations below those that induce
formation of the alternative dauer larva stage, suggesting that
exposure to pheromones can alter the chemosensory behaviors of
non-dauer animals. In addition, ASI expression of
srd-1
,
but not
str-2
and
str-3
, is induced by
sensory activity of the ASI neurons. Expression of two receptor genes
is regulated by developmental entry into the dauer larva stage.
srd-1
expression in ASI neurons is repressed in dauer
larvae.
str-2
expression in dauer animals is induced in
the ASI neurons, but repressed in the AWC neurons. The ASI and AWC
neurons remodel in the dauer stage, and these results suggest that
their sensory specificity changes as well. We suggest that
experience-dependent changes in chemosensory receptor gene expression
may modify olfactory behaviors.
Hydration of the peptide backbone largely defines the thermodynamic propensity scale of residues at the C′ position of the C-capping box of α-helices
Authors: Thomas Susan T. , Loladze Vakhtang V. , Makhatadze George I.
Published: July 2002

The C′ position of the C-capping box is the second residue outside of the helix. Statistical analysis of residue distribution at the C′ position in the α-helices' C-capping box showed that different amino acid residues occur with different probabilities, with the strongest preference being for glycine. To understand the physico-chemical basis for this preference, we studied the effects that 17 amino acid substitutions at the C′ position in an α-helix of ubiquitin have on the stability of this protein. We determined the following rank order of amino acid residues at the C′ position with respect to their effect on the stability: Gly>His>Asn>Arg>Lys>Gln>Ala>Phe>Met>Ser>Asp>Glu>Trp>Thr>Pro>Ile>Val. The effect of the amino acid substitutions on the structure also was evaluated by comparing the
1
H-
15
N heteronuclear sequential quantum correlation spectra and showed no significant changes in the structures of the most stable (Gly) and the least stable (Val) variants. The obtained changes in stability highly correlate (
r
= 0.85) with the statistical distribution of the residues at the C′ position indicating that the measured thermodynamic propensities are unbiased by secondary interactions. We also found that the measured thermodynamic propensities correlate well with the amide hydrogen exchange data on short model peptides (
r
= 0.85) and the calculated hydration of the peptide backbone (
r
= 0.88). These results combined with the changes in enthalpy and entropy of unfolding of ubiquitin variants suggest that dehydration of the peptide backbone plays a significant role in defining the thermodynamic propensity scale at the C′ position of the C-capping box in α-helices. This propensity scale is useful for protein secondary structure predictions and protein design.
Atmospheric aerosols as prebiotic chemical reactors
Authors: Dobson Christopher M. , Ellison G. Barney , Tuck Adrian F. , Vaida Veronica
Published: July 2002

Aerosol particles in the atmosphere have recently been found to
contain a large number of chemical elements and a high content of
organic material. The latter property is explicable by an inverted
micelle model. The aerosol sizes with significant atmospheric lifetimes
are the same as those of single-celled organisms, and they are
predicted by the interplay of aerodynamic drag, surface tension, and
gravity. We propose that large populations of such aerosols could have
afforded an environment, by means of their ability to concentrate
molecules in a wide variety of physical conditions, for key chemical
transformations in the prebiotic world. We also suggest that aerosols
could have been precursors to life, since it is generally agreed that
the common ancestor of terrestrial life was a single-celled organism.
The early steps in some of these initial transformations should be
accessible to experimental investigation.
Surface molecular recognition
Authors: Sampson Nicole S. , Mrksich Milan , Bertozzi Carolyn R.
Published: July 2002

The spatial display of cellular ligands and receptors is important for cell adhesion and communication. Two approaches that emphasize developing selective methods to dissect, modify, and control receptor–ligand interactions at the cellular interface are discussed.
Conformation-family Monte Carlo: A new method for crystal structure prediction
Authors: Pillardy Jaroslaw , Arnautova Yelena A. , Czaplewski Cezary , Gibson Kenneth D. , Scheraga Harold A.
Published: July 2002

A new global optimization method, Conformation-family Monte Carlo, has been developed recently for searching the conformational space of macromolecules. In the present paper, we adapted this method for prediction of crystal structures of organic molecules without assuming any symmetry constraints except the number of molecules in the unit cell. This method maintains a database of low energy structures that are clustered into families. The structures in this database are improved iteratively by a Metropolis-type Monte Carlo procedure together with energy minimization, in which the search is biased toward the regions of the lowest energy families. The Conformation-family Monte Carlo method is applied to a set of nine rigid and flexible organic molecules by using two popular force fields, AMBER and W99. The method performed well for the rigid molecules and reasonably well for the molecules with torsional degrees of freedom.
What is a moment? “Cortical” sensory integration over a brief interval
Authors: Hopfield J. J. , Brody Carlos D.
Published: July 2002

Recognition of complex temporal sequences is a general sensory
problem that requires integration of information over time. We describe
a very simple “organism” that performs this task, exemplified
here by recognition of spoken monosyllables. The network's computation
can be understood through the application of simple but generally
unexploited principles describing neural activity. The organism is a
network of very simple neurons and synapses; the experiments are
simulations. The network's recognition capabilities are robust to
variations across speakers, simple masking noises, and large variations
in system parameters. The network principles underlying recognition of
short temporal sequences are applied here to speech, but similar ideas
can be applied to aspects of vision, touch, and olfaction. In this
article, we describe only properties of the system that could be
measured if it were a real biological organism. We delay publication of
the principles behind the network's operation as an intellectual
challenge: the essential principles of operation can be deduced based
on the experimental results presented here alone. An interactive web
site (
http://neuron.princeton.edu/∼moment
) is available to
allow readers to design and carry out their own experiments on the
organism.
Importance of the carboxyl-terminal domain of enzyme I of the Escherichia coli phosphoenolpyruvate: sugar phosphotransferase system for phosphoryl donor specificity.
Authors: Seok Y J , Lee B R , Zhu P P , Peterkofsky A
Published: July 2002

The first protein component of the Escherichia coli phosphoenolpyruvate: sugar phosphotransferase system (PTS) is the 64-kDa protein enzyme I (EI), which can be phosphorylated by phosphoenolpyruvate (PEP) and carry out phosphotransfer to the acceptor heat-stable protein (HPr). The isolated amino-terminal domain (EIN) of E. coli EI is no longer phosphorylated by PEP but retains the ability to participate in reversible phosphotransfer to HPr. An expression vector was constructed for the production of large amounts of EIN, and conditions were developed for maximal expression of the protein. A three-column procedure is described for purification to homogeneity of EIN; a 500-ml culture yields approximately 80 mg of pure protein in about a 75% yield. Intact E. coli EI is effective in phosphotransfer from PEP to HPr from E. coli but not to the HPrs from Bacillus subtilis or Mycoplasma capricolum. Phosphotransfer from EI to enzyme IIAglc (EIIAglc) from E. coli or M. capricolum requires the intermediacy of HPr. The phosphorylated form of EIN is capable of more general phosphotransfer; it will effect phosphotransfer to HPrs from E. coli, B. subtilis, and M. capricolum as well as to EIAglc from E. coli. These studies demonstrate that the carboxyl-terminal domain of EI confers on the protein the capability to accept a phosphoryl group from PEP as well as a discriminator function that allows the intact protein to promote effective phosphoryl transfer only to E. coli HPr.
Irreversible inactivation of interleukin 2 in a pump-based delivery environment.
Authors: Tzannis S T , Hrushesky W J , Wood P A , Przybycien T M
Published: July 2002

The physical stability of pharmaceutical proteins in delivery environments is a critical determinant of biological potency and treatment efficacy, and yet it is often taken for granted. We studied both the bioactivity and physical stability of interleukin 2 upon delivery via continuous infusion. We found that the biological activity of the delivered protein was dramatically reduced by approximately 90% after a 24-hr infusion program. Only a portion of these losses could be attributed to direct protein deposition on the delivery surfaces. Analysis of delivered protein by size exclusion chromatography gave no indication of insulin-like, surface-induced aggregation phenomena. Examination of the secondary and tertiary structure of both adsorbed and delivered protein via Fourier-transform infrared spectroscopy, circular dichroism, and fluorescence spectroscopy indicated that transient surface association of interleukin 2 with the catheter tubing resulted in profound, irreversible structural changes that were responsible for the majority of the biological activity losses.
Two mitochondrial group I introns in a metazoan, the sea anemone Metridium senile: one intron contains genes for subunits 1 and 3 of NADH dehydrogenase.
Authors: Beagley C T , Okada N A , Wolstenholme D R
Published: July 2002

Mitochondrial genes for cytochrome c oxidase subunit I (COI) and NADH dehydrogenase subunit 5 (ND5) of the sea anemone Metridium senile (phylum Cnidaria) each contain a group I intron. This is in contrast to the reported absence of introns in all other metazoan mtDNAs so far examined. The ND5 intron is unusual in that it ends with A and contains two genes (ND1 and ND3) encoding additional subunits of NADH dehydrogenase. Correctly excised ND5 introns are not circularized but are precisely cleaved near their 3' ends and polyadenylylated to provide bicistronic transcripts of ND1 and ND3. COI introns, which encode a putative homing endonuclease, circularize, but in a way that retains the entire genome-encoded intron sequence (other group I introns are circularized with loss of a short segment of the intron 5' end). Introns were detected in the COI and ND5 genes of other sea anemones, but not in the COI and ND5 genes of other cnidarians. This suggests that the sea anemone mitochondrial introns may have been acquired relatively recently.
Presynaptic association of Rad51 protein with selected sites in meiotic chromatin.
Authors: Plug A W , Xu J , Reddy G , Golub E I , Ashley T
Published: July 2002

Eukaryotic homologs of Escherichia coli Rec-A protein have been shown to form nucleoprotein filaments with single-stranded DNA that recognize homologous sequences in duplex DNA. Several recent reports in four widely diverse species have demonstrated the association of RecA homologs with meiotic prophase chromatin. The current immunocytological study on mouse spermatocytes and oocytes shows that a eukaryotic homolog, Rad5l, associates with a subset of chromatin sites as early as premeiotic S phase, hours before either the appearance of precursors of synaptonemal complexes or the initiation of synapsis. When homologous chromosomes do begin to pair, the Rad5l-associated sequences are sites of initial contact between homologues and of localized DNA synthesis. Distribution of Rad5l foci on the chromatin of fully synapsed bivalents at early pachynema corresponds to an R-band pattern of mitotic chromosomes. R-bands are known to be preferred sites of both synaptic initiation and recombination. The time course of appearance of Rad51 association with chromatin, its distribution, and its interaction with other Rad5l-associated sequences suggests that it plays an important role preselection of sequences and synaptic initiation.
Radiation target analysis of RNA.
Authors: Benstein S L , Kempner E
Published: July 2002

Ribozymes are polynucleotide molecules with intrinsic catalytic activity, capable of cleaving nucleic acid substrates. Large RNA molecules were synthesized containing a hammerhead ribozyme moiety of 52 nucleotides linked to an inactive leader sequence, for total lengths of either 262 or 1226 nucleotides. Frozen RNAs were irradiated with high energy electrons. Surviving ribozyme activity was determined using the ability of the irradiated ribozymes to cleave a labeled substrate. The amount of intact RNA remaining was determined from the same irradiated samples by scanning the RNA band following denaturing gel electrophoresis. Radiation target analyses of these data revealed a structural target size of 80 kDa and a ribozyme activity target size of 15 kDa for the smaller ribozyme, and 319 kDa and 16 kDa, respectively, for the larger ribozyme. The disparity in target size for activity versus structure indicates that, in contrast to proteins, there is no spread of radiation damage far from the primary site of ionization in RNA molecules. The smaller target size for activity indicates that only primary ionizations occurring in the specific active region are effective. This is similar to the case for oligosaccharides. We concluded that the presence of the ribose sugar in the polymer chain restricts radiation damage to a small region and prevents major energy transfer throughout the molecule. Radiation target analysis should be a useful technique for evaluating local RNA:RNA and RNA:protein interactions in vitro.
A three-hybrid system to detect RNA-protein interactions in vivo.
Authors: SenGupta D J , Zhang B , Kraemer B , Pochart P , Fields S , Wickens M
Published: July 2002

RNA-protein interactions are pivotal in fundamental cellular processes such as translation, mRNA processing, early development, and infection by RNA viruses. However, in spite of the central importance of these interactions, few approaches are available to analyze them rapidly in vivo. We describe a yeast genetic method to detect and analyze RNA-protein interactions in which the binding of a bifunctional RNA to each of two hybrid proteins activates transcription of a reporter gene in vivo. We demonstrate that this three-hybrid system enables the rapid, phenotypic detection of specific RNA-protein interactions. As examples, we use the binding of the iron regulatory protein 1 (IRP1) to the iron response element (IRE), and of HIV trans-activator protein (Tat) to the HIV trans-activation response element (TAR) RNA sequence. The three-hybrid assay we describe relies only on the physical properties of the RNA and protein, and not on their natural biological activities; as a result, it may have broad application in the identification of RNA-binding proteins and RNAs, as well as in the detailed analysis of their interactions.
Doxycycline-mediated quantitative and tissue-specific control of gene expression in transgenic mice.
Authors: Kistner A , Gossen M , Zimmermann F , Jerecic J , Ullmer C , Lübbert H , Bujard H
Published: July 2002

The tet regulatory system in which doxycycline (dox) acts as an inducer of specifically engineered RNA polymerase II promoters was transferred into transgenic mice. Tight control and a broad range of regulation spanning up to five orders of magnitude were monitored dependent on the dox concentration in the water supply of the animals. Administration of dox rapidly induces the synthesis of the indicator enzyme luciferase whose activity rises over several orders of magnitude within the first 4 h in some organs. Induction is complete after 24 h in most organs analyzed. A comparable regulatory potential was revealed with the tet regulatory system where dox prevents transcription activation. Directing the synthesis of the tetracycline-controlled transactivator (tTA) to the liver led to highly specific regulation in hepatocytes where, in presence of dox, less than one molecule of luciferase was detected per cell. By contrast, a more than 10(5)-fold activation of the luciferase gene was observed in the absence of the antibiotic. This regulation was homogeneous throughout but stringently restricted to hepatocytes. These results demonstrate that both tetracycline-controlled transcriptional activation systems provide genetic switches that permit the quantitative control of gene activities in transgenic mice in a tissue-specific manner and, thus, suggest possibilities for the generation of a novel type of conditional mutants.
Estradiol stimulates tyrosine phosphorylation of the insulin-like growth factor-1 receptor and insulin receptor substrate-1 in the uterus.
Authors: Richards R G , DiAugustine R P , Petrusz P , Clark G C , Sebastian J
Published: July 2002

The signaling pathways associated with estrogen-induced proliferation of epithelial cells in the reproductive tract have not been defined. To identify receptor tyrosine kinases that are activated in vivo by 17 beta-estradiol (E2), uteri from ovariectomized mice were examined for enhanced tyrosine phosphorylation of various receptors and a receptor substrate following treatment with this hormone. Within 4 hr after hormone exposure, extracts showed increased phosphotyrosine (P-Tyr) immunoreactivity at several bands, including 170- and 180-kDa; these bands were still apparent at 24 hr after E2. Analysis of immunoprecipitates from uterine extracts revealed that E2 enhanced tyrosine phosphorylation of the insulin-like growth factor-1 receptor (IGF-1R) and insulin receptor substrate-1 (IRS-1) by 6 hr. Comparison of supernatants from IRS-1 and control rabbit IgG immunoprecipitates indicated that the 170-kDa P-Tyr band in extracts was equivalent to IRS-1. The receptors for epidermal growth factor, platelet-derived growth factor, and basic fibroblast growth factor did not exhibit an E2-induced increase in P-Tyr content. The nonestrogenic steroid hormones examined did not stimulate the P-Tyr content of IGF-1R or IRS-1. Immunolocalization of P-Tyr and IRS-1 revealed strong reactivity in the epithelial layer of the uterus from E2-treated mice, suggesting that the majority of P-Tyr bands observed in immunoblots originate in the epithelium. Since hormonal activation of IRS-1 is epithelial, estrogen-specific, and initiated before maximal DNA synthesis occurs following treatment with hormone, this protein, as part of the IGF-1R pathway, may be important in mediating estrogen-stimulated proliferation in the uterus.
Intron insertion facilitates amplification of cloned virus cDNA in Escherichia coli while biological activity is reestablished after transcription in vivo.
Authors: Johansen I E
Published: July 2002

Insertion of introns into cloned cDNA of two isolates of the plant potyvirus pea seedborne mosaic virus facilitated plasmid amplification in Escherichia coli. Multiple stop codons in the inserted introns interrupted the open reading frame of the virus cDNA, thereby terminating undesired translation of virus proteins in E. coli. Plasmids containing the full-length virus sequences, placed under control of the cauliflower mosaic virus 35S promoter and the nopaline synthase termination signal, were stable and easy to amplify in E. coli if one or more introns were inserted into the virus sequence. These plasmids were infectious when inoculated mechanically onto Pisum sativum leaves. Examination of the cDNA-derived viruses confirmed that intron splicing of in vivo transcribed pre-mRNA had occurred as predicted, reestablishing the virus genome sequences. Symptom development and virus accumulation of the cDNA derived viruses and parental viruses were identical. It is proposed that intron insertion can be used to facilitate manipulation and amplification of cloned DNA fragments that are unstable in, or toxic to, E. coli. When transcribed in vivo in eukaryotic cells, the introns will be eliminated from the sequence and will not interfere with further analysis of protein expression or virus infection.
Evidence that the 42- and 40-amino acid forms of amyloid β protein are generated from the β-amyloid precursor protein by different protease activities
Authors: Citron Martin , Diehl Thekla S. , Gordon Grace , Biere Anja Leona , Seubert Peter , Selkoe Dennis J.
Published: July 2002

Cerebral deposition of the amyloid β protein (Aβ) is an early
and invariant feature of Alzheimer disease (AD). Whereas the 40-amino
acid form of Aβ (Aβ
40
) accounts for ≈90% of all Aβ
normally released from cells, it appears to contribute only to later
phases of the pathology. In contrast, the longer more amyloidogenic
42-residue form (Aβ
42
), accounting for only ≈10% of
secreted Aβ, is deposited in the earliest phase of AD and remains the
major constituent of most amyloid plaques throughout the disease.
Moreover, its levels have been shown to be increased in all known forms
of early-onset familial AD. Thus, inhibition of Aβ
42
production is a prime therapeutic goal. The same protease,
γ-secretase, is assumed to generate the C termini of both
Aβ
40
and Aβ
42
. Herein, we analyze the
effect of the compound MDL 28170, previously suggested to inhibit
γ-secretase, on β-amyloid precursor protein processing. By
immunoprecipitating conditioned medium of different cell lines with
various Aβ
40
- and Aβ
42
-specific antibodies,
we demonstrate a much stronger inhibition of the γ-secretase cleavage
at residue 40 than of that at residue 42. These data suggest that
different proteases generate the Aβ
40
and
Aβ
42
C termini. Further, they raise the possibility of
identifying compounds that do not interfere with general β-amyloid
precursor protein metabolism, including Aβ
40
production,
but specifically block the generation of the pathogenic
Aβ
42
peptide.
Memory from the dynamics of intrinsic membrane currents
Authors: Marder Eve , Abbott L. F. , Turrigiano Gina G. , Liu Zheng , Golowasch Jorge
Published: July 2002

Almost all theoretical and experimental studies of the mechanisms
underlying learning and memory focus on synaptic efficacy and make the
implicit assumption that changes in synaptic efficacy are both
necessary and sufficient to account for learning and memory. However,
network dynamics depends on the complex interaction between intrinsic
membrane properties and synaptic strengths and time courses.
Furthermore, neuronal activity itself modifies not only synaptic
efficacy but also the intrinsic membrane properties of neurons. This
paper presents examples demonstrating that neurons with complex
temporal dynamics can provide short-term “memory” mechanisms that
rely solely on intrinsic neuronal properties. Additionally, we discuss
the potential role that activity may play in long-term modification of
intrinsic neuronal properties. While not replacing synaptic plasticity
as a powerful learning mechanism, these examples suggest that memory in
networks results from an ongoing interplay between changes in synaptic
efficacy and intrinsic membrane properties.
An abundantly expressed mucin-like protein from Toxocara canis infective larvae: the precursor of the larval surface coat glycoproteins.
Authors: Gems D , Maizels R M
Published: July 2002

Evasion of host immunity by Toxocara canis infective larvae is mediated by the nematode surface coat, which is shed in response to binding by host antibody molecules or effector cells. The major constituent of the coat is the TES-120 glycoprotein series. We have isolated a 730-bp cDNA from the gene encoding the apoprotein precursor of TES-120. The mRNA is absent from T. canis adults but hyperabundant in larvae, making up approximately 10% of total mRNA, and is trans-spliced with the nematode 5' leader sequence SL1. It encodes a 15.8-kDa protein (after signal peptide removal) containing a typical mucin domain: 86 amino acid residues, 72.1% of which are Ser or Thr, organized into an array of heptameric repeats, interspersed with proline residues. At the C-terminal end of the putative protein are two 36-amino acid repeats containing six Cys residues, in a motif that can also be identified in several genes in Caenorhabditis elegans. Although TES-120 displays size and charge heterogeneity, there is a single copy gene and a homogeneous size of mRNA. The association of overexpression of some membrane-associated mucins with immunosuppression and tumor metastasis suggests a possible model for the role of the surface coat in immune evasion by parasitic nematodes.