bluuebunny/crossref_metadata_2025 huggingface dataset:
Shifts in the life history of parasitic wasps correlate with pronounced alterations in early development
Authors: Grbic Miodrag , Strand Michael R.
Published: July 2002

Developmental processes have been traditionally viewed to be invariant within higher taxa. However, examples are known whereby closely related species exhibit alterations in early embryogenesis yet appear very similar as adults. Such developmental changes are thought to occur in response to shifts in life history. In insects, the regulation of embryonic development has been intensively studied in model species like
Drosophila melanogaster
. Previous comparative studies suggest that the developmental processes documented in
Drosophila
well describe embryogenesis of advanced, holometabolous, insects generally. There have been few attempts, however, to take into account how life history has influenced early development of insects or to characterize early development of species with life histories fundamentally different from flies. Here we compared early development of two species from the same family of parasitic wasps that exhibit very different life histories.
Bracon hebetor
is an ectoparasite that lays large, yolky eggs on the integument of its host that develop much like the free-living honeybee and
Drosophila
. In contrast,
Aphidius ervi
is an endoparasite that lays small and apparently yolk-free eggs that develop in the hemocoel of the host. This wasp exhibits a radically different mode of early development at both the cellular and molecular level from
B. hebetor
. The developmental changes in
A. ervi
reflect functional adaptations for its derived life history and argue that departures from the fly paradigm may occur commonly among insects whose eggs develop under conditions different from typical terrestrial species.
Behind the scenes of functional brain imaging: A historical and physiological perspective
Authors: Raichle Marcus E.
Published: July 2002

At the forefront of cognitive neuroscience research in normal
humans are the new techniques of functional brain imaging: positron
emission tomography and magnetic resonance imaging. The signal used by
positron emission tomography is based on the fact that changes in the
cellular activity of the brain of normal, awake humans and laboratory
animals are accompanied almost invariably by changes in local blood
flow. This robust, empirical relationship has fascinated scientists for
well over a hundred years. Because the changes in blood flow are
accompanied by lesser changes in oxygen consumption, local changes in
brain oxygen content occur at the sites of activation and provide the
basis for the signal used by magnetic resonance imaging. The biological
basis for these signals is now an area of intense research stimulated
by the interest in these tools for cognitive neuroscience research.
β-
d
-Glucosyl-hydroxymethyluracil is a conserved DNA modification in kinetoplastid protozoans and is abundant in their telomeres
Authors: van Leeuwen Fred , Taylor Martin C. , Mondragon Angeles , Moreau Hervé , Gibson Wendy , Kieft Rudo , Borst Piet
Published: July 2002

The unusual DNA base β-
d
-glucosyl-hydroxymethyluracil, called “J,” replaces ≈0.5–1% of Thy in DNA of African trypanosomes but has not been found in other organisms thus far. In
Trypanosoma brucei
, J is located predominantly in repetitive DNA, and its presence correlates with the silencing of telomeric genes. Using antibodies specific for J, we have developed sensitive assays to screen for J in a range of organisms and have found that J is not limited to trypanosomes that undergo antigenic variation but is conserved among Kinetoplastida. In all kinetoplastids tested, including the human pathogens
Leishmania donovani
and
Trypanosoma cruzi
, J was found to be abundantly present in the (GGGTTA)
n
telomere repeats. Outside Kinetoplastida, J was found only in
Diplonema
, a small phagotrophic marine flagellate, in which we also identified 5-MeCyt. Fractionation of
Diplonema
DNA showed that the two modifications are present in a common genome compartment, which suggests that they may have a similar function. Dinoflagellates appear to contain small amounts of modified bases that may be analogs of J. The evolutionary conservation of J in kinetoplastid protozoans suggests that it has a general function, repression of transcription or recombination, or a combination of both.
T. brucei
may have recruited J for the control of genes involved in antigenic variation.
Activating mutations in the extracellular domain of the fibroblast growth factor receptor 2 function by disruption of the disulfide bond in the third immunoglobulin-like domain
Authors: Robertson Scott C. , Meyer April N. , Hart Kristen C. , Galvin Brendan D. , Webster Melanie K. , Donoghue Daniel J.
Published: July 2002

Multiple human skeletal and craniosynostosis disorders, including Crouzon, Pfeiffer, Jackson–Weiss, and Apert syndromes, result from numerous point mutations in the extracellular region of fibroblast growth factor receptor 2 (FGFR2). Many of these mutations create a free cysteine residue that potentially leads to abnormal disulfide bond formation and receptor activation; however, for noncysteine mutations, the mechanism of receptor activation remains unclear. We examined the effect of two of these mutations, W290G and T341P, on receptor dimerization and activation. These mutations resulted in cellular transformation when expressed as FGFR2/Neu chimeric receptors. Additionally, in full-length FGFR2, the mutations induced receptor dimerization and elevated levels of tyrosine kinase activity. Interestingly, transformation by the chimeric receptors, dimerization, and enhanced kinase activity were all abolished if either the W290G or the T341P mutation was expressed in conjunction with mutations that eliminate the disulfide bond in the third immunoglobulin-like domain (Ig-3). These results demonstrate a requirement for the Ig-3 cysteine residues in the activation of FGFR2 by noncysteine mutations. Molecular modeling also reveals that noncysteine mutations may activate FGFR2 by altering the conformation of the Ig-3 domain near the disulfide bond, preventing the formation of an intramolecular bond. This allows the unbonded cysteine residues to participate in intermolecular disulfide bonding, resulting in constitutive activation of the receptor.
Detection and characterization of carcinoma cells in the blood
Authors: Racila Emilian , Euhus David , Weiss Arthur J. , Rao Chandra , McConnell John , Terstappen Leon W. M. M. , Uhr Jonathan W.
Published: July 2002

A highly sensitive assay combining immunomagnetic enrichment with multiparameter flow cytometric and immunocytochemical analysis has been developed to detect, enumerate, and characterize carcinoma cells in the blood. The assay can detect one epithelial cell or less in 1 ml of blood. Peripheral blood (10–20 ml) from 30 patients with carcinoma of the breast, from 3 patients with prostate cancer, and from 13 controls was examined by flow cytometry for the presence of circulating epithelial cells defined as nucleic acid
+
, CD45
−
, and cytokeratin
+
. Highly significant differences in the number of circulating epithelial cells were found between normal controls and patients with cancer including 17 with organ-confined disease. To determine whether the circulating epithelial cells in the cancer patients were neoplastic cells, cytospin preparations were made after immunomagnetic enrichment and were analyzed. Epithelial cells from patients with breast cancer generally stained with mAbs against cytokeratin and 3 of 5 for mucin-1. In contrast, no cells that stained for these antigens were observed in the blood from normal controls. The morphology of the stained cells was consistent with that of neoplastic cells. Of 8 patients with breast cancer followed for 1–10 months, there was a good correlation between changes in the level of tumor cells in the blood with both treatment with chemotherapy and clinical status. The present assay may be helpful in early detection, in monitoring disease, and in prognostication.
Neurons in the dorsal column white matter of the spinal cord: Complex neuropil in an unexpected location
Authors: Abbadie Catherine , Skinner Kate , Mitrovic Igor , Basbaum Allan I.
Published: July 2002

It is common to think of gray matter as the site of integration in neural circuits and white matter as the wires that connect different groups of neurons. The dorsal column (DC) white matter, for example, is the spinal cord axonal pathway through which a topographic map of the body is conveyed to the somatosensory cortex. We now describe a network of neurons located along the midline of the DCs. The neurons are present in several mammals, including primates and birds, and have a profuse dendritic arbor that expresses both the neuron-specific marker, microtubule-associated protein-2, and the neurokinin-1 receptor, a target of the neuropeptide, substance P. Electron microscopy and double immunostaining for synaptophysin and a marker of γ-aminobutyric acid-ergic terminals documented a rich synaptic input to these neurons. Finally, injection of a γ-aminobutyric acid type A receptor antagonist or of substance P into the cerebrospinal fluid of the rat spinal cord induced Fos expression and internalization of the neurokinin-1 receptor in these neurons, respectively, indicating that the DC neurons are under tonic inhibitory control and can respond to neurotransmitters that circulate in the cerebrospinal fluid.
The cyclin D1 gene is a target of the β-catenin/LEF-1 pathway
Authors: Shtutman Michael , Zhurinsky Jacob , Simcha Inbal , Albanese Chris , D’Amico Mark , Pestell Richard , Ben-Ze’ev Avri
Published: July 2002

β-Catenin plays a dual role in the cell: one in linking the cytoplasmic side of cadherin-mediated cell–cell contacts to the actin cytoskeleton and an additional role in signaling that involves transactivation in complex with transcription factors of the lymphoid enhancing factor (LEF-1) family. Elevated β-catenin levels in colorectal cancer caused by mutations in β-catenin or by the adenomatous polyposis coli molecule, which regulates β-catenin degradation, result in the binding of β-catenin to LEF-1 and increased transcriptional activation of mostly unknown target genes. Here, we show that the cyclin D1 gene is a direct target for transactivation by the β-catenin/LEF-1 pathway through a LEF-1 binding site in the cyclin D1 promoter. Inhibitors of β-catenin activation, wild-type adenomatous polyposis coli, axin, and the cytoplasmic tail of cadherin suppressed cyclin D1 promoter activity in colon cancer cells. Cyclin D1 protein levels were induced by β-catenin overexpression and reduced in cells overexpressing the cadherin cytoplasmic domain. Increased β-catenin levels may thus promote neoplastic conversion by triggering cyclin D1 gene expression and, consequently, uncontrolled progression into the cell cycle.
An imprinted, mammalian bicistronic transcript encodes two independent proteins
Authors: Gray Todd A. , Saitoh Shinji , Nicholls Robert D.
Published: July 2002

Polycistronic transcripts are common in prokaryotes but rare in eukaryotes. Phylogenetic analysis of the
SNRPN
(SmN) mRNA in five eutherian mammals reveals a second highly conserved coding sequence, termed
SNURF
(
SNRPN u
pstream
r
eading
f
rame). The vast majority of nucleotide substitutions in
SNURF
occur in the wobble codon position, providing strong evolutionary evidence for selection for protein-coding function. Because
SNURF–SNRPN
maps to human chromosome 15q11–q13 and is paternally expressed, each cistron is a candidate for a role in the imprinted Prader-Willi syndrome (PWS) and PWS mouse models.
SNURF
encodes a highly basic 71-aa protein that is nuclear-localized (as is SmN). Because
SNURF
is the only protein-coding sequence within the imprinting regulatory region in 15q11–q13, it may have provided the original selection for imprinting in this domain. Whereas some human tissues express a minor
SNURF
-only transcript, mouse tissues express only the bicistronic
Snurf–Snrpn
transcript. We show that both
SNURF
and
SNRPN
are translated in normal, but not PWS, human, and mouse tissues and cell lines. These findings identify SNURF as a protein that is produced along with SmN from a bicistronic transcript; polycistronic mRNAs therefore are encoded in mammalian genomes where they may form functional operons.
World food trends and prospects to 2025
Authors: Dyson Tim
Published: July 2002

This paper reviews food (especially cereal) production trends and
prospects for the world and its main regions. Despite fears to the
contrary, in recent years we have seen continued progress toward better
methods of feeding humanity. Sub-Saharan Africa is the sole major
exception. Looking to the future, this paper argues that the
continuation of recent cereal yield trends should be sufficient to cope
with most of the demographically driven expansion of cereal demand that
will occur until the year 2025. However, because of an increasing
degree of mismatch between the expansion of regional demand and the
potential for supply, there will be a major expansion of world cereal
(and noncereal food) trade. Other consequences for global agriculture
arising from demographic growth include the need to use water much
more efficiently and an even greater dependence on nitrogen fertilizers
(e.g., South Asia). Farming everywhere will depend more on
information-intensive agricultural management procedures. Moreover,
despite continued general progress, there still will be a significant
number of undernourished people in 2025. Signs of heightened harvest
variability, especially in North America, are of serious concern. Thus,
although future general food trends are likely to be positive, in some
respects we also could be entering a more volatile world.
Heme transfer to the heme chaperone CcmE during cytochrome
c
maturation requires the CcmC protein, which may function independently of the ABC-transporter CcmAB
Authors: Schulz Henk , Fabianek Renata A. , Pellicioli Erica C. , Hennecke Hauke , Thöny-Meyer Linda
Published: July 2002

Cytochrome
c
maturation in
Escherichia coli
requires the
ccm
operon, which encodes eight membrane proteins (CcmABCDEFGH). CcmE is a periplasmic heme chaperone that binds heme covalently and transfers it onto apocytochrome
c
in the presence of CcmF, CcmG, and CcmH. In this work we addressed the functions of the
ccmABCD
gene products with respect to holo-CcmE formation and the subsequent ligation of heme to apocytochrome
c
. In the absence of the
ccmABCD
genes, heme is not bound to CcmE. We report that CcmC is functionally uncoupled from the ABC transporter subunits CcmA and CcmB, because it is the only Ccm protein that is strictly required for heme transfer and attachment to CcmE. Site-directed mutagenesis of conserved histidines inactivates the CcmC protein, which is in agreement with the hypothesis that this protein interacts directly with heme. We also present evidence that questions the role of CcmAB as a heme exporter; yet, the transported substrate remains unknown. CcmD was found to be involved in stabilizing the heme chaperone CcmE in the membrane. We propose a heme-trafficking pathway as part of a substantially revised model for cytochrome
c
maturation in
E. coli
.
Cation-π interactions in structural biology
Authors: Gallivan Justin P. , Dougherty Dennis A.
Published: July 2002

Cation-π interactions in protein structures are identified and evaluated by using an energy-based criterion for selecting significant sidechain pairs. Cation-π interactions are found to be common among structures in the Protein Data Bank, and it is clearly demonstrated that, when a cationic sidechain (Lys or Arg) is near an aromatic sidechain (Phe, Tyr, or Trp), the geometry is biased toward one that would experience a favorable cation-π interaction. The sidechain of Arg is more likely than that of Lys to be in a cation-π interaction. Among the aromatics, a strong bias toward Trp is clear, such that over one-fourth of all tryptophans in the data bank experience an energetically significant cation-π interaction.
Retroviral vectors preloaded with a viral receptor-ligand bridge protein are targeted to specific cell types
Authors: Boerger Adrienne L. , Snitkovsky Sophie , Young John A. T.
Published: July 2002

Successful targeting methods represent a major hurdle to the use of retroviral vectors in cell-specific gene-delivery applications. We recently described an approach for retroviral targeting with a retroviral receptor-ligand bridge protein that was bound to the cognate cell-surface ligand receptors before viral challenge. We now report a significant improvement made to this viral targeting method by using a related bridge protein, designated TVB-EGF, comprised of the extracellular domain of the TVB receptor for subgroup B avian leukosis virus fused to epidermal growth factor (EGF). The most important activity of TVB-EGF was that it allowed specific viral entry when preloaded onto virions. Furthermore, virions preloaded with TVB-EGF were thermostable and could be produced directly from virus- packaging cells. These data suggest an approach for targeting retroviral vectors to specific cell types by using virions preloaded with a retroviral receptor-ligand bridge protein and indicate that these types of bridge proteins may be useful reagents for studying the normal mechanism of retroviral entry.
Epoxomicin, a potent and selective proteasome inhibitor, exhibitsin vivoantiinflammatory activity
Authors: Meng Lihao , Mohan Royce , Kwok Benjamin H. B. , Elofsson Mikael , Sin Ny , Crews Craig M.
Published: July 2002

The proteasome regulates cellular processes as diverse as cell cycle progression and NF-κB activation. In this study, we show that the potent antitumor natural product epoxomicin specifically targets the proteasome. Utilizing biotinylated-epoxomicin as a molecular probe, we demonstrate that epoxomicin covalently binds to the LMP7, X, MECL1, and Z catalytic subunits of the proteasome. Enzymatic analyses with purified bovine erythrocyte proteasome reveal that epoxomicin potently inhibits primarily the chymotrypsin-like activity. The trypsin-like and peptidyl-glutamyl peptide hydrolyzing catalytic activities also are inhibited at 100- and 1,000-fold slower rates, respectively. In contrast to peptide aldehyde proteasome inhibitors, epoxomicin does not inhibit nonproteasomal proteases such trypsin, chymotrypsin, papain, calpain, and cathepsin B at concentrations of up to 50 μM. In addition, epoxomicin is a more potent inhibitor of the chymotrypsin-like activity than lactacystin and the peptide vinyl sulfone NLVS. Epoxomicin also effectively inhibits NF-κB activation in vitro and potently blocksin vivoinflammation in the murine ear edema assay. These results thus define epoxomicin as a novel proteasome inhibitor that likely will prove useful in exploring the role of the proteasome in variousin vivoandin vitrosystems.
Antitumor activity of mannan-binding proteinin vivoas revealed by a virus expression system: Mannan-binding proteindependent cell-mediated cytotoxicity
Authors: Ma Yong , Uemura Kazuhide , Oka Shogo , Kozutsumi Yasunori , Kawasaki Nobuko , Kawasaki Toshisuke
Published: July 2002

Mannan-binding protein (MBP), a Ca2+-dependent mammalian lectin specific for mannose andN-acetylglucosamine, is an important serum component associated with innate immunity. MBP activates complement and functions as a direct opsonin on binding to mannooligosaccharide-bearing pathogens. We have found that MBP recognizes and binds specifically to oligosaccharide ligands expressed on the surfaces of a human colorectal carcinoma. Interestingly, the recombinant vaccinia virus carrying human MBP gene was demonstrated to possess a potent growth-inhibiting activity against human colorectal carcinoma cells transplanted in KSN nude mice when administered by intratumoral or subcutaneous injection. Moreover, a significant prolongation of life span of tumor-bearing mice resulted from the treatment. This effect appears to be a consequence of local production of MBP. Unexpectedly, the mutant MBP, which had essentially no complement-activating activity, was nearly as active as wild-type MBP. These results indicated that MBP has a previously undescribed cytotoxic activity, which we propose to term MBP-dependent cell-mediated cytotoxicity(MDCC). In addition, this study provides a model for the development of an effective and specific host defense factor for cancer gene therapy.
Nonstatistical binding of a protein to clustered carbohydrates
Authors: Horan Nina , Yan Lin , Isobe Hiroyuki , Whitesides George M. , Kahne Daniel
Published: July 2002

Carbohydrate-derivatized self-assembled monolayers (SAMs) are used as a model system to address issues involving cell-surface carbohydrate–protein interactions. Here we examine the influence of carbohydrate surface density on protein-binding avidity. We show that the binding selectivity of
Bauhinia purpurea
lectin switches from one carbohydrate ligand to another as the surface density of the carbohydrate ligands increases from values of χ
sugar
≈ 0.1–1.0. Polyvalent binding is possible at all surface densities investigated; hence, the switch in selectivity is not due simply to the achievement of a critical density that permits polyvalent contacts. Instead, secondary interactions at high surface densities promote a switch in carbohydrate-binding selectivity. These findings may have implications for how changes in the composition and the density of cell-surface carbohydrates influence biological recognition processes and regulatory pathways.
Tropomyosin directly modulates actomyosin mechanical performance at the level of a single actin filament
Authors: VanBuren Peter , Palmiter Kimberly A. , Warshaw David M.
Published: July 2002

Muscle contraction is the result of myosin cross-bridges (XBs) cyclically interacting with the actin-containing thin filament. This interaction is modulated by the thin filament regulatory proteins, troponin and tropomyosin (Tm). With the use of an
in vitro
motility assay, the role of Tm in myosin’s ability to generate force and motion was assessed. At saturating myosin surface densities, Tm had no effect on thin filament velocity. However, below 50% myosin saturation, a significant reduction in actin–Tm filament velocity was observed, with complete inhibition of movement occurring at 12.5% of saturating surface densities. Under similar conditions, actin filaments alone demonstrated no reduction in velocity. The effect of Tm on force generation was assessed at the level of a single thin filament. In the absence of Tm, isometric force was a linear function of the density of myosin on the motility surface. At 50% myosin surface saturation, the presence of Tm resulted in a 2-fold enhancement of force relative to actin alone. However, no further potentiation of force was observed with Tm at saturating myosin surface densities. These results indicate that, in the presence of Tm, the strong binding of myosin cooperatively activates the thin filament. The inhibition of velocity at low myosin densities and the potentiation of force at higher myosin densities suggest that Tm can directly modulate the kinetics of a single myosin XB and the recruitment of a population of XBs, respectively. At saturating myosin conditions, Tm does not appear to affect the recruitment or the kinetics of myosin XBs.
Exploring the origins of topological frustration: Design of a minimally frustrated model of fragment B of protein A
Authors: Shea Joan-Emma , Onuchic José N. , Brooks Charles L.
Published: July 2002

Topological frustration in an energetically unfrustrated off-lattice model of the helical protein fragment B of protein A from
Staphylococcus aureus
was investigated. This Gō-type model exhibited thermodynamic and kinetic signatures of a well-designed two-state folder with concurrent collapse and folding transitions and single exponential kinetics at the transition temperature. Topological frustration is determined in the absence of energetic frustration by the distribution of Fersht φ values. Topologically unfrustrated systems present a unimodal distribution sharply peaked at intermediate φ, whereas highly frustrated systems display a bimodal distribution peaked at low and high φ values. The distribution of φ values in protein A was determined both thermodynamically and kinetically. Both methods yielded a unimodal distribution centered at φ = 0.3 with tails extending to low and high φ values, indicating the presence of a small amount of topological frustration. The contacts with high φ values were located in the turn regions between helices I and II and II and III, intimating that these hairpins are in large part required in the transition state. Our results are in good agreement with all-atom simulations of protein A, as well as lattice simulations of a three- letter code 27-mer (which can be compared with a 60-residue helical protein). The relatively broad unimodal distribution of φ values obtained from the all-atom simulations and that from the minimalist model for the same native fold suggest that the structure of the transition state ensemble is determined mostly by the protein topology and not energetic frustration.
Steroid disorders in children: Congenital adrenal hyperplasia and apparent mineralocorticoid excess
Authors: New Maria I. , Wilson Robert C.
Published: July 2002

Our research team and laboratories have concentrated on two inherited endocrine disorders, congenital adrenal hyperplasia (CAH) and apparent mineralocorticoid excess, in thier investigations of the pathophysiology of adrenal steroid hormone disorders in children. CAH refers to a family of inherited disorders in which defects occur in one of the enzymatic steps required to synthesize cortisol from cholesterol in the adrenal gland. Because of the impaired cortisol secretion, adrenocorticotropic hormone levels rise due to impairment of a negative feedback system, which results in hyperplasia of the adrenal cortex. The majority of cases is due to 21-hydroxylase deficiency (21-OHD). Owing to the blocked enzymatic step, cortisol precursors accumulate in excess and are converted to potent androgens, which are secreted and cause
in utero
virilization of the affected female fetus genitalia in the classical form of CAH. A mild form of the 21-OHD, termed nonclassical 21-OHD, is the most common autosomal recessive disorder in humans, and occurs in 1/27 Ashkenazic Jews. Mutations in the
CYP21
gene have been identified that cause both classical and nonclassical CAH. Apparent mineralocorticoid excess is a potentially fatal genetic disorder causing severe juvenile hypertension, pre- and postnatal growth failure, and low to undetectable levels of potassium, renin, and aldosterone. It is caused by autosomal recessive mutations in the
HSD11B2
gene, which result in a deficiency of 11β-hydroxysteroid dehydrogenase type 2. In 1998, we reported a mild form of this disease, which may represent an important cause of low-renin hypertension.
A conserved residue in the tip proteins of CS1 and CFA/I pili of enterotoxigenic
Escherichia coli
that is essential for adherence
Authors: Sakellaris Harry , Munson George P. , Scott June R.
Published: July 2002

Enterotoxigenic
Escherichia coli
associated with human diarrheal disease utilize any of a limited group of serologically distinguishable pili for attachment to intestinal cells. These include CS1 and CFA/I pili. We show here that chemical modification of arginyl residues in CS1 pili abolishes CS1-mediated agglutination of bovine erythrocytes, which serves as a model system for attachment. Alanine substitution of the single arginyl residue in CooA, the major pilin, had no effect on the assembly of pili or on hemagglutination. In contrast, substitution of alanine for R181 in CooD, the minor pilin associated with the pilus tip, abolished hemagglutination, and substitution of R20 reduced hemagglutination. Neither of these substitutions affected CS1 pilus assembly. This shows that CooD is essential for CS1-mediated attachment and identifies specific residues that are involved in receptor binding but not in pilus assembly. In addition to mediating agglutination of bovine erythrocytes, CFA/I also mediates agglutination of human erythrocytes. Substitution of R181 by alanine in the CooD homolog, CfaE, abolished both of these reactions. We conclude that the same region of the pilus tip protein is involved in adherence of CS1 and CFA/I pili, although their receptor specificities differ. This suggests that the region of the pilus tip adhesin protein that includes R181 might be an appropriate target for therapeutic intervention or for a vaccine to protect against human diarrhea caused by enterotoxigenic
E. coli
strains that have serologically different pili.
INAF, a protein required for transient receptor potential Ca
2+
channel function
Authors: Li Chenjian , Geng Chaoxian , Leung Hung-Tat , Hong Young Seok , Strong Lydia L. R. , Schneuwly Stephan , Pak William L.
Published: July 2002

The
trp
gene of
Drosophila
encodes a subunit of a class of Ca
2+
-selective light-activated channels that carry the bulk of the phototransduction current. Transient receptor potential (TRP) homologs have been identified throughout animal phylogeny. In vertebrates, TRP-related channels have been suggested to mediate “store-operated Ca
2+
entry,” which is important in Ca
2+
homeostasis in a wide variety of cell types. However, the mechanisms of activation and regulation of the TRP channel are not known. Here, we report on the
Drosophila inaF
gene, which encodes a highly eye-enriched protein, INAF, that appears to be required for TRP channel function. A null mutation in this gene significantly reduces the amount of the TRP protein and, in addition, specifically affects the TRP channel function so as to nearly shut down its activity. The
inaF
mutation also dramatically suppresses the severe degeneration caused by a constitutively active mutation in the
trp
gene. Although the reduction in the amount of the TRP protein may contribute to these phenotypes, several lines of evidence support the view that
inaF
mutations also more directly affect the TRP channel function, suggesting that the INAF protein may have a regulatory role in the channel function.
Semaphorin 3A growth cone collapse requires a sequence homologous to tarantula hanatoxin
Authors: Behar Oded , Mizuno Keiko , Badminton Mike , Woolf Clifford J.
Published: July 2002

Axonal guidance is key to the formation of neuronal circuitry. Semaphorin 3A (Sema 3A; previously known as semaphorin III, semaphorin D, and collapsin-1), a secreted subtype of the semaphorin family, is an important axonal guidance molecule
in vitro
and
in vivo
. The molecular mechanisms of the repellent activity of semaphorins are, however, poorly understood. We have now found that the secreted semaphorins contain a short sequence of high homology to hanatoxin, a tarantula K
+
and Ca
2+
ion channel blocker. Point mutations in the hanatoxin-like sequence of Sema 3A reduce its capacity to repel embryonic dorsal root ganglion axons. Sema 3A growth cone collapse activity is inhibited by hanatoxin, general Ca
2+
channel blockers, a reduction in extracellular or intracellular Ca
2+
, and a calmodulin inhibitor, but not by K
+
channel blockers. Our data support an important role for Ca
2+
in mediating the Sema 3A response and suggest that Sema 3A may produce its effects by causing the opening of Ca
2+
channels.
Antitumor activity and pharmacokinetics of a mixed-backbone antisense oligonucleotide targeted to the RIα subunit of protein kinase A after oral administration
Authors: Wang Hui , Cai Qiuyin , Zeng Xiaofei , Yu Dong , Agrawal Sudhir , Zhang Ruiwen
Published: July 2002

Overexpression of the RIα subunit of cAMP-dependent protein kinase (PKA) has been demonstrated in various human cancers. PKA has been suggested as a potential target for cancer therapy. The goal of the present study was to evaluate an anti-PKA antisense oligonucleotide (mixed-backbone oligonucleotide) as a therapeutic approach to human cancer treatment. The identified oligonucleotide inhibited the growth of cell lines of human colon cancer (LS174T, DLD-1), leukemia (HL-60), breast cancer (MCF-7, MDA-MB-468), and lung cancer (A549) in a time-, concentration-, and sequence-dependent manner. In a dose-dependent manner, the oligonucleotide displayed
in vivo
antitumor activity in severe combined immunodeficient and nude mice bearing xenografts of human cancers of the colon (LS174T), breast (MDA-MB-468), and lung (A549). The routes of drug administration were intraperitoneal and oral. Synergistic effects were found when the antisense oligonucleotide was used in combination with the cancer chemotherapeutic agent cisplatin. The pharmacokinetics of the oligonucleotide after oral administration of
35
S-labeled oligonucleotide into tumor-bearing mice indicated an accumulation and retention of the oligonucleotide in tumor tissue. This study further provides a basis for clinical studies of the antisense oligonucleotide targeted to the RI
α
subunit of PKA (GEM 231) as a cancer therapeutic agent used alone or in combination with conventional chemotherapy.
An alternative pathway for rod signals in the rodent retina: Rod photoreceptors, cone bipolar cells, and the localization of glutamate receptors
Authors: Hack Iris , Peichl Leo , Brandstätter Johann Helmut
Published: July 2002

In the mammalian retina, extensive processing of spatiotemporal and chromatic information occurs. One key principle in signal transfer through the retina is parallel processing. Two of these parallel pathways are the ON- and OFF-channels transmitting light and dark signals. This dual system is created in the outer plexiform layer, the first relay station in retinal signal transfer. Photoreceptors release glutamate onto ON- and OFF-type bipolar cells, which are functionally distinguished by their postsynaptic expression of different types of glutamate receptors, namely ionotropic and metabotropic glutamate receptors. In the current concept, rod photoreceptors connect only to rod bipolar cells (ON-type) and cone photoreceptors connect only to cone bipolar cells (ON- and OFF-type). We have studied the distribution of (
RS
)-α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) glutamate receptor subunits at the synapses in the outer plexiform layer of the rodent retina by immunoelectron microscopy and serial section reconstruction. We report a non-classical synaptic contact and an alternative pathway for rod signals in the retina. Rod photoreceptors made synaptic contact with putative OFF-cone bipolar cells that expressed the AMPA glutamate receptor subunits GluR1 and GluR2 on their dendrites. Thus, in the retina of mouse and rat, an alternative pathway for rod signals exists, where rod photoreceptors bypass the rod bipolar cell and directly excite OFF-cone bipolar cells through an ionotropic sign-conserving AMPA glutamate receptor.
Cloning of chlorophyllase, the key enzyme in chlorophyll degradation: Finding of a lipase motif and the induction by methyl jasmonate
Authors: Tsuchiya Tohru , Ohta Hiroyuki , Okawa Katsuya , Iwamatsu Akihiro , Shimada Hiroshi , Masuda Tatsuru , Takamiya Ken-ichiro
Published: July 2002

Chlorophyllase (Chlase) is the first enzyme involved in chlorophyll (Chl) degradation and catalyzes the hydrolysis of ester bond to yield chlorophyllide and phytol. In the present study, we isolated the Chlase cDNA. We synthesized degenerate oligo DNA probes based on the internal amino acid sequences of purified Chlase fromChenopodium album, screened theC. albumcDNA library, and cloned a cDNA (CaCLH,C. albumchlorophyll-chlorophyllido hydrolase). The deduced amino acid sequence (347 aa residues) had a lipase motif overlapping with an ATP/GTP-binding motif (P-loop).CaCLH possibly was localized in the extraplastidic part of the cell, because a putative signal sequence for endoplasmic reticulum is at the N terminus. The amino acid sequence shared 37% identity with a function-unknown gene whose mRNA is inducible by coronatine and methyl jasmonate (MeJA) inArabidopsis thaliana(AtCLH1). We expressed the gene products ofAtCLH1and ofCaCLHinEscherichia coli, and they similarly exhibited Chlase activity. Moreover, we isolated another full-length cDNA based on anArabidopsisgenomic fragment and expressed it inE. coli, demonstrating the presence of the secondArabidopsisCLH gene (AtCLH2). No typical feature of signal sequence was identified inAtCLH1, whereasAtCLH2 had a typical signal sequence for chloroplast.AtCLH1mRNA was induced rapidly by a treatment of MeJA, which is known to promote senescence and Chl degradation in plants, and a high mRNA level was maintained up to 9 h.AtCLH2, however, did not respond to MeJA.
Surface geochemistry of the clay minerals
Authors: Sposito Garrison , Skipper Neal T. , Sutton Rebecca , Park Sung-ho , Soper Alan K. , Greathouse Jeffery A.
Published: July 2002

Clay minerals are layer type aluminosilicates that figure in
terrestrial biogeochemical cycles, in the buffering capacity of the
oceans, and in the containment of toxic waste materials. They are also
used as lubricants in petroleum extraction and as industrial catalysts
for the synthesis of many organic compounds. These applications derive
fundamentally from the colloidal size and permanent structural charge
of clay mineral particles, which endow them with significant surface
reactivity. Unraveling the surface geochemistry of hydrated clay
minerals is an abiding, if difficult, topic in earth sciences research.
Recent experimental and computational studies that take advantage of
new methodologies and basic insights derived from the study of
concentrated ionic solutions have begun to clarify the structure of
electrical double layers formed on hydrated clay mineral surfaces,
particularly those in the interlayer region of swelling 2:1 layer type
clay minerals. One emerging trend is that the coordination of
interlayer cations with water molecules and clay mineral surface
oxygens is governed largely by cation size and charge, similarly to a
concentrated ionic solution, but the location of structural charge
within a clay layer and the existence of hydrophobic patches on its
surface provide important modulations. The larger the interlayer
cation, the greater the influence of clay mineral structure and
hydrophobicity on the configurations of adsorbed water molecules. This
picture extends readily to hydrophobic molecules adsorbed within an
interlayer region, with important implications for clay–hydrocarbon
interactions and the design of catalysts for organic synthesis.
Maize as a model for the evolution of plant nuclear genomes
Authors: Gaut Brandon S. , Le Thierry d'Ennequin Maud , Peek Andrew S. , Sawkins Mark C.
Published: July 2002

The maize genome is replete with chromosomal duplications and
repetitive DNA. The duplications resulted from an ancient polyploid
event that occurred over 11 million years ago. Based on DNA sequence
data, the polyploid event occurred after the divergence between sorghum
and maize, and hence the polyploid event explains some of the
difference in DNA content between these two species. Genomic
rearrangement and diploidization followed the polyploid event. Most of
the repetitive DNA in the maize genome is retrotransposable elements,
and they comprise 50% of the genome. Retrotransposon multiplication
has been relatively recent—within the last 5–6 million
years—suggesting that the proliferation of retrotransposons has also
contributed to differences in DNA content between sorghum and maize.
There are still unanswered questions about repetitive DNA, including
the distribution of repetitive DNA throughout the genome, the relative
impacts of retrotransposons and chromosomal duplication in plant genome
evolution, and the hypothesized correlation of duplication events with
transposition. Population genetic processes also affect the evolution
of genomes. We discuss how centromeric genes should, in theory, contain
less genetic diversity than noncentromeric genes. In addition, studies
of diversity in the wild relatives of maize indicate that different
genes have different histories and also show that domestication and
intensive breeding have had heterogeneous effects on genetic diversity
across genes.
Structure-based discovery of an organic compound that binds Bcl-2 protein and induces apoptosis of tumor cells
Authors: Wang Jia-Lun , Liu Dongxiang , Zhang Zhi-Jia , Shan Simei , Han Xiaobing , Srinivasula Srinivasa M. , Croce Carlo M. , Alnemri Emad S. , Huang Ziwei
Published: July 2002

Bcl-2 and related proteins are key regulators of apoptosis or programmed cell death implicated in human disease including cancer. We recently showed that cell-permeable Bcl-2 binding peptides could induce apoptosis of human myeloid leukemia
in vitro
and suppress its growth in severe combined immunodeficient mice. Here we report the discovery of HA14-1, a small molecule (molecular weight = 409) and nonpeptidic ligand of a Bcl-2 surface pocket, by using a computer screening strategy based on the predicted structure of Bcl-2 protein.
In vitro
binding studies demonstrated the interaction of HA14-1 with this Bcl-2 surface pocket that is essential for Bcl-2 biological function. HA14-1 effectively induced apoptosis of human acute myeloid leukemia (HL-60) cells overexpressing Bcl-2 protein that was associated with the decrease in mitochondrial membrane potential and activation of caspase-9 followed by caspase-3. Cytokine response modifier A, a potent inhibitor of Fas-mediated apoptosis, did not block apoptosis induced by HA14-1. Whereas HA14-1 strongly induced the death of NIH 3T3 (Apaf-1
+/+
) cells, it had little apoptotic effect on Apaf-1-deficient (Apaf-1
−/−
) mouse embryonic fibroblast cells. These data are consistent with a mechanism by which HA14-1 induces the activation of Apaf-1 and caspases, possibly by binding to Bcl-2 protein and inhibiting its function. The discovery of this cell-permeable molecule provides a chemical probe to study Bcl-2-regulated apoptotic pathways
in vivo
and could lead to the development of new therapeutic agents.
Reactive oxygen and nitrogen intermediates in the relationship between mammalian hosts and microbial pathogens
Authors: Nathan Carl , Shiloh Michael U.
Published: July 2002

This review summarizes recent evidence from knock-out mice on the role of reactive oxygen intermediates and reactive nitrogen intermediates (RNI) in mammalian immunity. Reflections on redundancy in immunity help explain an apparent paradox: the phagocyte oxidase and inducible nitric oxide synthase are each nonredundant, and yet also mutually redundant, in host defense. In combination, the contribution of these two enzymes appears to be greater than previously appreciated. The remainder of this review focuses on a relatively new field, the basis of microbial resistance to RNI. Experimental tuberculosis provides an important example of an extended, dynamic balance between host and pathogen in which RNI play a major role. In diseases such as tuberculosis, a molecular understanding of host–pathogen interactions requires characterization of the defenses used by microbes against RNI, analogous to our understanding of defenses against reactive oxygen intermediates. Genetic and biochemical approaches have identified candidates for RNI-resistance genes inMycobacterium tuberculosisand other pathogens.
The N terminus of microsomal Δ9 stearoyl-CoA desaturase contains the sequence determinant for its rapid degradation
Authors: Mziaut Hassan , Korza George , Ozols Juris
Published: July 2002

Stearoyl-CoA desaturase (SCD) is a key regulator of membrane fluidity, turns over rapidly, and represents a model for selective degradation of short-lived proteins of the endoplasmic reticulum (ER). The mechanism whereby specific ER proteins are targeted for degradation in the midst of stable proteins coexisting in the same membrane is unknown. To investigate the intracellular fate of SCD and to identify the determinants involved in the rapid turnover of SCD, we created chimeras of SCD tagged at the C terminus with the green fluorescent protein (GFP). The fusion proteins were expressed in Chinese hamster ovary cells and exhibited an ER localization. Unlike native GFP, the SCD–GFP construct was unstable and had a half life of a few hours. Truncated fusion proteins consisting of residues 27–358 and 45–358 of SCD linked to the N terminus of GFP were stable. To investigate the general applicability of the N terminus of SCD in the destabilization of proteins, we fused residues 1–33 of SCD to the N terminus of GFP. The resulting chimera was extremely short lived. To investigate the effect of membrane sidedness on the fusion protein degradation, we attached a lumenal targeting signal to the N terminus of SCD 1–33-GFP. The construct was localized to the lumen of ER and was metabolically stable, indicating that SCD degradation signal functions on the cytosolic rather than the lumenal side of the ER. These results demonstrate that the N-terminal segment of some 30 residues of SCD constitutes a motif responsible for the rapid degradation of SCD.
How well can an amoeba climb?
Authors: Fukui Yoshio , Uyeda Taro Q. P. , Kitayama Chikako , Inoué Shinya
Published: July 2002

We report here our efforts to measure the crawling force generated by cells undergoing amoeboid locomotion. In a centrifuge microscope, acceleration was increased until amoebae of
Dictyostelium discoideum
were “stalled” or no longer able to “climb up.” The “apparent weight” of the amoebae at stalling rpm in myosin mutants depended on the presence of myosin II (but not myosins IA and IB) and paralleled the cortical strength of the cells. Surprisingly, however, the cell stalled not only in low-density media as expected but also in media with densities greater than the cell density where the buoyant force should push the amoeba upward. We find that the leading pseudopod is bent under centrifugal force in all stalled amoebae, suggesting that this pseudopod is very dense indeed. This finding also suggests that directional cell locomotion against resistive forces requires a turgid forward-pointing pseudopod, most likely sustained by cortical actomyosin II.
Crystal structures of bovine milk xanthine dehydrogenase and xanthine oxidase: Structure-based mechanism of conversion
Authors: Enroth Cristofer , Eger Bryan T. , Okamoto Ken , Nishino Tomoko , Nishino Takeshi , Pai Emil F.
Published: July 2002

Mammalian xanthine oxidoreductases, which catalyze the last two steps in the formation of urate, are synthesized as the dehydrogenase form xanthine dehydrogenase (XDH) but can be readily converted to the oxidase form xanthine oxidase (XO) by oxidation of sulfhydryl residues or by proteolysis. Here, we present the crystal structure of the dimeric (Mr, 290,000) bovine milk XDH at 2.1-Å resolution and XO at 2.5-Å resolution and describe the major changes that occur on the proteolytic transformation of XDH to the XO form. Each molecule is composed of an N-terminal 20-kDa domain containing two iron sulfur centers, a central 40-kDa flavin adenine dinucleotide domain, and a C-terminal 85-kDa molybdopterin-binding domain with the four redox centers aligned in an almost linear fashion. Cleavage of surface-exposed loops of XDH causes major structural rearrangement of another loop close to the flavin ring (Gln 423—Lys 433). This movement partially blocks access of the NAD substrate to the flavin adenine dinucleotide cofactor and changes the electrostatic environment of the active site, reflecting the switch of substrate specificity observed for the two forms of this enzyme.