bluuebunny/crossref_metadata_2025 huggingface dataset:
A role of Hsp60 in autoimmune diabetes: analysis in a transgenic model.
Authors: Birk O S , Douek D C , Elias D , Takacs K , Dewchand H , Gur S L , Walker M D , van der Zee R , Cohen I R , Altmann D M
Published: July 2002

A pathogenic role for self-reactive cells against the stress protein Hsp60 has been proposed as one of the events leading to autoimmune destruction of pancreatic beta cells in the diabetes of nonobese diabetic (NOD) mice. To examine this hypothesis, we generated transgenic NOD mice carrying a murine Hsp60 transgene driven by the H-2E alpha class II promoter. This would be expected to direct expression of the transgene to antigen-presenting cells including those in the thymus and so induce immunological tolerance by deletion. Detailed analysis of Hsp60 expression revealed that the endogenous gene is itself expressed strongly in thymic medullary epithelium (and weakly in cortex) yet fails to induce tolerance. Transgenic mice with retargeted Hsp60 showed overexpression of the gene in thymic cortical epithelium and in bone marrow-derived cells. Analysis of spontaneous T-cell responses to a panel of self and heterologous Hsp60 antigens showed that tolerance to the protein had not been induced, although responses to an immunodominant 437-460 epitope implicated in disease were suppressed, probably indicating an epitope shift. This correlated with changes in disease susceptibility: insulitis in transgenic mice was substantially reduced so that pathology rarely progressed beyond periislet infiltration. This was reflected in a substantial reduction in hyperglycemia and disease. These data indicate that T cells specific for some epitopes of murine Hsp60 are likely to be involved in the islet-cell destruction that occurs in NOD mice.
Origin and evolution of circular waves and spirals in Dictyostelium discoideum territories.
Authors: Pálsson E , Cox E C
Published: July 2002

Randomly distributed Dictyostelium discoideum cells form cooperative territories by signaling to each other with cAMP. Cells initiate the process by sending out pulsatile signals, which propagate as waves. With time, circular and spiral patterns form. We show that by adding spatial and temporal noise to the levels of an important regulator of external cAMP levels, the cAMP phosphodiesterase inhibitor, we can explain the natural progression of the system from randomly firing cells to circular waves whose symmetries break to form double- and single- or multi-armed spirals. When phosphodiesterase inhibitor is increased with time, mimicking experimental data, the wavelength of the spirals shortens, and a proportion of them evolve into pairs of connected spirals. We compare these results to recent experiments, finding that the temporal and spatial correspondence between experiment and model is very close.
The response regulator SprE controls the stability of RpoS.
Authors: Pratt L A , Silhavy T J
Published: July 2002

In Escherichia coli, the sigma factor, RpoS, is a central regulator in stationary-phase cells. We have identified a gene, sprE (stationary-phase regulator), as essential for the negative regulation of rpoS expression. SprE negatively regulates the rpoS gene product at the level of protein stability, perhaps in response to nutrient availability. The ability of SprE to destabilize RpoS is dependent on the ClpX/ClpP protease. Based on homology, SprE is a member of the response regulator family of proteins. SprE is the first response regulator identified that is implicated in the control of protein stability. Moreover, SprE is the first reported protein that appears to regulate rpoS in response to a specific environmental parameter.
Site-specific de-
N
-glycosylation of diglycosylated ovalbumin in hen oviduct by endogenous peptide:
N
-glycanase as a quality control system for newly synthesized proteins
Authors: Suzuki Tadashi , Kitajima Ken , Emori Yasufumi , Inoue Yasuo , Inoue Sadako
Published: July 2002

Hen ovalbumin (OVA) is known to exist as a singly
N
-glycosylated form with a glycan chain on Asn-292 in egg white. Previous studies showed that di-
N
-glycosylated form of OVA [Di-OVA; CHO-Asn-292/CHO-Asn-311 (CHO,
N
-glycan chain)], which has two
N
-glycan chains on Asn-292 and Asn-311, was expressed only transiently in hen oviduct. Di-OVA was not found in egg white, suggesting that this form cannot be secreted normally and may possibly be converted to mono-
N
-glycosylated OVA (CHO-Asn-292/Asp-311) by the action of peptide:
N
-glycanase (PNGase) during synthesis and secretion. In this study, we have identified the putative PNGase activity in the homogenate of hen oviduct, purified 1,000-fold, and designated as PNGase HO. We examined the reactivity of Di-OVA to PNGase HO and found that this enzyme site-specifically cleaved off the glycan chain at Asn-311 to convert Di-OVA into the mono-
N
-glycosylated form (CHO-Asn-292/Asp-311). In contrast, this enzyme was found not to act on the mono-
N
-glycosylated OVA (CHO-Asn-292/Asn-311) found in egg white when it was tested as a substrate. The present findings support our view that de-
N
-glycosylation catalyzed by PNGase may be involved in quality control of newly synthesized proteins by converting its diglycosylated form into the mono-
N
-glycosylated form that can be secreted. However, the alternative possibility that de-
N
-glycosylation may trigger cytosolic degradation of the aberrantly glycosylated glycoprotein cannot be ruled out.
Ciliary neurotrophic factor corrects obesity and diabetes associated with leptin deficiency and resistance
Authors: Gloaguen Isabelle , Costa Patrizia , Demartis Anna , Lazzaro Domenico , Di Marco Annalise , Graziani Rita , Paonessa Giacomo , Chen Fang , Rosenblum Charles I. , Van der Ploeg Lex H. T. , Cortese Riccardo , Ciliberto Gennaro , Laufer Ralph
Published: July 2002

Receptor subunits for the neurocytokine ciliary neurotrophic factor (CNTF) share sequence similarity with the receptor for leptin, an adipocyte-derived cytokine involved in body weight homeostasis. We report here that CNTF and leptin activate a similar pattern of STAT factors in neuronal cells, and that mRNAs for CNTF receptor subunits, similarly to the mRNA of leptin receptor, are localized in mouse hypothalamic nuclei involved in the regulation of energy balance. Systemic administration of CNTF or leptin led to rapid induction of the
tis-11
primary response gene in the arcuate nucleus, suggesting that both cytokines can signal to hypothalamic satiety centers. Consistent with this idea, CNTF treatment of
ob
/
ob
mice, which lack functional leptin, was found to reduce the adiposity, hyperphagia, and hyperinsulinemia associated with leptin deficiency. Unlike leptin, CNTF also reduced obesity-related phenotypes in
db
/
db
mice, which lack functional leptin receptor, and in mice with diet-induced obesity, which are partially resistant to the actions of leptin. The identification of a cytokine-mediated anti-obesity mechanism that acts independently of the leptin system may help to develop strategies for the treatment of obesity associated with leptin resistance.
Growth arrest of Epstein–Barr virus immortalized B lymphocytes by adenovirus-delivered ribozymes
Authors: Huang Shuang , Stupack Dwayne , Mathias Patricia , Wang Yibing , Nemerow Glen
Published: July 2002

Epstein–Barr virus (EBV) infection is associated with several human diseases that involve unrestricted proliferation of B lymphocytes. EBV nuclear antigen 1 (EBNA-1) is expressed in all EBV-infected cells and plays an essential role in persistence of the EBV genome. EBNA-1 has also been reported to have oncogenic potential. As an approach for treating EBV infections, we examined the capacity of EBNA-1 ribozymes delivered by recombinant adenoviruses to suppress EBNA-1 expression and to block virus-induced B cell proliferation. In contrast to primary B cells, EBV-transformed B lymphoblastoid cell lines expressed αv integrins, the adenovirus internalization receptors, and were also susceptible to adenovirus-mediated gene delivery. Adenovirus delivery of a specific ribozyme (RZ1) to lymphoblastoid cell lines, suppressed EBNA-1 mRNA and protein expression, significantly reduced the number of EBV genomes, and nearly abolished cell proliferation in low serum. Adenovirus delivery of RZ1 also prevented EBV infection of an established EBV-negative B cell line. These studies demonstrate the potential use of adenovirus-encoded ribozymes to treat EBV-induced lymphoproliferative disorders.
A novel role for clathrin in cytokinesis
Authors: Niswonger Maria L. , O’Halloran Theresa J.
Published: July 2002

Using clathrin-minus
Dictyostelium
cells, we identified a novel requirement for clathrin during cytokinesis. In suspension culture, clathrin-minus cells failed to divide and became large and multinucleate. This cytokinesis deficiency was not attributable to a pleiotropic effect on the actomyosin cytoskeleton, since other cellular events driven by myosin II (e.g., cortical contraction and capping of concanavalin A receptors) remained intact in clathrin-minus cells. Examination of cells expressing myosin II tagged with green fluorescent protein showed that clathrin-minus cells failed to assemble myosin II into a functional contractile ring. This inability to localize myosin II to a particular location was specific for cytokinesis, since clathrin-minus cells moving across a substrate localized myosin II properly to their posterior cortexes. These results demonstrate that clathrin is essential for construction of a functional contractile ring during cell division.
Epithelial antibiotic induced in states of disease
Authors: Stolzenberg Ethan D. , Anderson G. Mark , Ackermann Mark R. , Whitlock Robert H. , Zasloff Michael
Published: July 2002

Epithelial defensins provide an active defense against the external microbial environment. We investigated the distribution and expression of this class of antimicrobial peptides in normal cattle and in animals in varying states of disease. β-defensin mRNA was found to be widely expressed in numerous exposed epithelia but was found at higher levels in tissues that are constantly exposed to and colonized by microorganisms. We observed induction in ileal mucosa during chronic infection with
Mycobacterium paratuberculosis
and in bronchial epithelium after acute infection with
Pasteurella haemolytica
. It has been proposed that expression of antimicrobial peptides is an integral component of the inflammatory response. The results reported here support this hypothesis and suggest that epithelial defensins provide a rapidly mobilized local defense against infectious organisms.
Autoradiographic characterization of [
3
H]-5-HT-moduline binding sites in rodent brain and their relationship to 5-HT
1B
receptors
Authors: Cloëz-Tayarani Isabelle , Cardona Ana , Rousselle Jean-Claude , Massot Olivier , Edelman Lena , Fillion Gilles
Published: July 2002

5-HT-moduline is an endogenous tetrapeptide [Leu-Ser-Ala-Leu (LSAL)] that was first isolated from bovine brain tissue. To understand the physiological role of this tetrapeptide, we studied the localization of 5-HT-moduline binding sites in rat and mouse brains. Quantitative data obtained with a gaseous detector of β-particles (β-imager) indicated that [
3
H]-5-HT-moduline bound specifically to rat brain sections with high affinity (
K
d
= 0.77 nM and
B
max
= 0.26 dpm/mm
2
). Using film autoradiography in parallel, we found that 5-HT-moduline binding sites were expressed in a variety of rat and mouse brain structures. In 5-HT
1B
receptor knock-out mice, the specific binding of [
3
H]-5-HT-moduline was not different from background labeling, indicating that 5-HT-moduline targets are exclusively located on the 5-HT
1B
receptors. Although the distribution of 5-HT-moduline binding sites was similar to that of 5-HT
1B
receptors, they did not overlap totally. Differences in distribution patterns were found in regions containing either high levels of 5-HT
1B
receptors such as globus pallidus and subiculum that were poorly labeled or in other regions such as dentate gyrus of hippocampus and cortex where the relative density of 5-HT-moduline binding sites was higher than that of 5-HT
1B
receptors. In conclusion, our data, based on autoradiographic localization, indicate that 5-HT-moduline targets are located on 5-HT
1B
receptors present both on 5-HT afferents and postsynaptic neurons. By interacting specifically with 5-HT
1B
receptors, this tetrapeptide may play a pivotal role in pathological states such as stress that involves the dysfunction of 5-HT neurotransmission.
Bacterial resistance to vancomycin: Overproduction, purification, and characterization of VanC2 from
Enterococcus casseliflavus
as a
d
-Ala-
d
-Ser ligase
Authors: Park Il-Seon , Lin Chun-Hung , Walsh Christopher T.
Published: July 2002

The VanC phenotype for clinical resistance of enterococci to
vancomycin is exhibited by
Enterococcus gallinarum
and
Enterococcus casseliflavus
. Based on the detection of
the cell precursor UDP-
N
-acetylmuramic acid pentapeptide
intermediate terminating in
d
-Ala-
d
-Ser
instead of
d
-Ala-
d
-Ala, it has been predicted
that the VanC ligase would be a
d
-Ala-
d
-Ser rather than a
d
-Ala-
d
-Ala ligase. Overproduction of the
E. casseliflavus
ATCC 25788
vanC2
gene in
Escherichia coli
and its purification to homogeneity
allowed demonstration of ATP-dependent
d
-Ala-
d
-Ser ligase activity. The
k
cat
/
K
m2
(
K
m2
=
K
m
for
d
-Ser or C-terminal
d
-Ala) ratio for
d
-Ala-
d
-Ser/
d
-Ala-
d
-Ala
dipeptide formation is 270/0.69 for a 400-fold selection against
d
-Ala in the C-terminal position. VanC2 also has
substantial
d
-Ala-
d
-Asn ligase activity
(
k
cat
/
K
m2
= 74
mM
−1
min
−1
).
Generation of CD8+T cells specific for transporter associated with antigen processing deficient cells
Authors: Wolpert Elisabeth Z. , Petersson Max , Chambers Benedict J. , Sandberg Johan K. , Kiessling Rolf , Ljunggren Hans-Gustaf , Kärre Klas
Published: July 2002

Cells with impaired transporter associated with antigen processing (TAP) function express low levels of cell surface major histocompatibility complex (MHC) class I molecules, and are generally resistant to lysis by MHC class I restricted cytotoxic T lymphocytes (CTLs). Here we report the generation of MHC class I restricted CD8+CTLs that surprisingly require target cell TAP deficiency for efficient recognition. C57BL/6 (B6) mice immunized with syngenic B7–1 (CD80) expressing TAP-deficient cells generated a potent CTL response against both TAP-deficient RMA-S tumor cells and TAP-deficient Con A blasts, whereas the corresponding TAP-expressing target cells were considerably less susceptible or resistant to lysis. The CTL epitopes recognized were expressed also by the human TAP-deficient cell line T2, transfected with appropriate MHC class I molecules. B6 mice immunized with B7–1-transfected TAP-deficient RMA-S cells were protected from outgrowth of a subsequent RMA-S tumor challenge. These findings are discussed in relation to the biochemical nature of MHC class I dependent CTL epitopes associated with impaired TAP function, as well as implications for immunotherapy and autoimmunity.
Decoding temporally encoded sensory input by cortical oscillations and thalamic phase comparators
Authors: Ahissar Ehud , Haidarliu Sebastian , Zacksenhouse Miriam
Published: July 2002

The temporally encoded information obtained by vibrissal touch
could be decoded “passively,” involving only input-driven elements,
or “actively,” utilizing intrinsically driven oscillators. A
previous study suggested that the trigeminal somatosensory system of
rats does not obey the bottom-up order of activation predicted by
passive decoding. Thus, we have tested whether this system obeys the
predictions of active decoding. We have studied cortical single units
in the somatosensory cortices of anesthetized rats and guinea pigs and
found that about a quarter of them exhibit clear spontaneous
oscillations, many of them around whisking frequencies (≈10 Hz). The
frequencies of these oscillations could be controlled locally by
glutamate. These oscillations could be forced to track the frequency of
induced rhythmic whisker movements at a stable, frequency-dependent,
phase difference. During these stimulations, the response intensities
of multiunits at the thalamic recipient layers of the cortex decreased,
and their latencies increased, with increasing input frequency. These
observations are consistent with thalamocortical loops implementing
phase-locked loops, circuits that are most efficient in decoding
temporally encoded information like that obtained by active vibrissal
touch. According to this model, and consistent with our results,
populations of thalamic “relay” neurons function as phase
“comparators” that compare cortical timing expectations with the
actual input timing and represent the difference by their population
output rate.
α-Tocopherol transfer protein stimulates the secretion of α-tocopherol from a cultured liver cell line through a brefeldin A-insensitive pathway
Authors: Arita Makoto , Nomura Kazuhiro , Arai Hiroyuki , Inoue Keizo
Published: July 2002

Vitamin E (α-tocopherol) is a fat-soluble antioxidant that is transported by plasma lipoproteins in the body. α-Tocopherol taken up by the liver with lipoprotein is thought to be resecreted into the plasma in very low density lipoprotein (VLDL). α-Tocopherol transfer protein (αTTP), which was recently identified as a product of the causative gene for familial isolated vitamin E deficiency, is a cytosolic liver protein and plays an important role in the efficient recycling of plasma vitamin E. To throw light on the mechanism of αTTP-mediated α-tocopherol transfer in the liver cell, we devised an assay system using the hepatoma cell line McARH7777. Using this system, we found that the secretion of α-tocopherol was more efficient in cells expressing αTTP than in matched cells lacking αTTP. Brefeldin A, which effectively inhibits VLDL secretion by disrupting the Golgi apparatus, had no effect on α-tocopherol secretion, indicating that αTTP-mediated α-tocopherol secretion is not coupled to VLDL secretion. Among other agents tested, only 25-hydroxycholesterol, a modulator of cholesterol metabolism, inhibited α-tocopherol secretion. This inhibition is most likely mediated by oxysterol-binding protein. These results suggest that αTTP present in the liver cytosol functions to stimulate secretion of cellular α-tocopherol into the extracellular medium and that the reaction utilizes a novel non-Golgi-mediated pathway that may be linked to cellular cholesterol metabolism and/or transport.
Phytosulfokine-α, a sulfated pentapeptide, stimulates the proliferation of rice cells by means of specific high- and low-affinity binding sites
Authors: Matsubayashi Yoshikatsu , Takagi Leiko , Sakagami Youji
Published: July 2002

Peptide growth factors were isolated from conditioned medium derived from rice (
Oryza sativa
L.) suspension cultures and identified to be a sulfated pentapeptide [H-Tyr(SO
3
H)-Ile-Tyr(SO
3
H)-Thr-Gln-OH] and its C-terminal-truncated tetrapeptide [H-Tyr(SO
3
H)-Ile-Tyr(SO
3
H)-Thr-OH]. These structures were identical to the phytosulfokines originally found in asparagus (
Asparagus officinalis
L.) mesophyll cultures. The pentapeptide [phytosulfokine-α (PSK-α)] very strongly stimulated colony formation of rice protoplasts at concentrations above 10
−8
M, indicating a similar mode of action in rice of phytosulfokines. Binding assays using
35
S-labeled PSK-α demonstrated the existence of both high- and low-affinity specific saturable binding sites on the surface of rice cells in suspension. Analysis of [
35
S]PSK-α binding in differential centrifugation fractions suggested association of the binding with a plasma membrane-enriched fraction. The apparent
K
d
values for [
35
S]PSK-α binding were found to be 1 × 10
−9
M for the high-affinity type and 1 × 10
−7
M for the low-affinity type, with maximal numbers of binding sites of 1 × 10
4
sites per cell and 1 × 10
5
sites per cell, respectively. Competition studies with [
35
S]PSK-α and several synthetic PSK-α analogs demonstrated that only peptides that possesses mitogenic activity can effectively displace the radioligand. These results suggest that a signal transduction pathway mediated by peptide factors is involved in plant cell proliferation.
Benthic–pelagic links and rocky intertidal communities: Bottom-up effects on top-down control?
Authors: Menge Bruce A. , Daley Bryon A. , Wheeler Patricia A. , Dahlhoff Elizabeth , Sanford Eric , Strub P. Ted
Published: July 2002

Insight into the dependence of benthic communities on biological and physical processes in nearshore pelagic environments, long considered a “black box,” has eluded ecologists. In rocky intertidal communities at Oregon coastal sites 80 km apart, differences in abundance of sessile invertebrates, herbivores, carnivores, and macrophytes in the low zone were not readily explained by local scale differences in hydrodynamic or physical conditions (wave forces, surge flow, or air temperature during low tide). Field experiments employing predator and herbivore manipulations and prey transplants suggested top-down (predation, grazing) processes varied positively with bottom-up processes (growth of filter-feeders, prey recruitment), but the basis for these differences was unknown. Shore-based sampling revealed that between-site differences were associated with nearshore oceanographic conditions, including phytoplankton concentration and productivity, particulates, and water temperature during upwelling. Further, samples taken at 19 sites along 380 km of coastline suggested that the differences documented between two sites reflect broader scale gradients of phytoplankton concentration. Among several alternative explanations, a coastal hydrodynamics hypothesis, reflecting mesoscale (tens to hundreds of kilometers) variation in the interaction between offshore currents and winds and continental shelf bathymetry, was inferred to be the primary underlying cause. Satellite imagery and offshore chlorophyll-
a
samples are consistent with the postulated mechanism. Our results suggest that benthic community dynamics can be coupled to pelagic ecosystems by both trophic and transport linkages.
Functional nonequality of the cardiac and skeletal ryanodine receptors
Authors: Nakai Junichi , Ogura Tatsuya , Protasi Feliciano , Franzini-Armstrong Clara , Allen Paul D. , Beam Kurt G.
Published: July 2002

Dihydropyridine receptors (DHPRs), which are voltage-gated Ca
2+
channels, and ryanodine receptors (RyRs), which are intracellular Ca
2+
release channels, are expressed in diverse cell types, including skeletal and cardiac muscle. In skeletal muscle, there appears to be reciprocal signaling between the skeletal isoforms of both the DHPR and the RyR (RyR-1), such that Ca
2+
release activity of RyR-1 is controlled by the DHPR and Ca
2+
channel activity of the DHPR is controlled by RyR-1. Dyspedic skeletal muscle cells, which do not express RyR-1, lack excitation–contraction coupling and have an ≈30-fold reduction in L-type Ca
2+
current density. Here we have examined the ability of the predominant cardiac and brain RyR isoform, RyR-2, to substitute for RyR-1 in interacting with the skeletal DHPR. When RyR-2 is expressed in dyspedic muscle cells, it gives rise to spontaneous intracellular Ca
2+
oscillations and supports Ca
2+
entry-induced Ca
2+
release. However, unlike RyR-1, the expressed RyR-2 does not increase the Ca
2+
channel activity of the DHPR, nor is the gating of RyR-2 controlled by the skeletal DHPR. Thus, the ability to participate in skeletal-type reciprocal signaling appears to be a unique feature of RyR-1.
Role of leptin in hypothalamic–pituitary function
Authors: Yu W. H. , Kimura M. , Walczewska A. , Karanth S. , McCann S. M.
Published: July 2002

A defect in the structure of the obese gene is responsible for development of obesity in the
ob
/
ob
mouse. The product of expression of the gene is the protein hormone leptin. Leptin causes weight loss in
ob
/
ob
and normal mice, it is secreted by adipocytes, and it is an important controller of the size of fat stores by inhibiting appetite. The
ob
/
ob
mouse is infertile and has a pattern of gonadotropin secretion similar to that of prepubertal animals. Consequently, we hypothesized that leptin might play a role in the control of gonadotropin secretion and initiated studies on its possible acute effects on hypothalamic–pituitary function. After a preincubation period, hemi-anterior pituitaries of adult male rats were incubated with leptin for 3 hr. Leptin produced a dose-related increase in follicle-stimulating hormone (FSH) and luteinizing hormone (LH) release, which reached peaks with 10
−9
and 10
−11
M leptin, respectively. Gonadotropin release decreased at higher concentrations of leptin to values indistinguishable from that of control pituitaries. On the other hand, prolactin secretion was greatly increased in a dose-related manner but only with leptin concentrations (10
−7
–10
−5
M). Incubation with leptin of median eminence–arcuate nuclear explants from the same animals produced significant increases in LH-releasing hormone (LHRH) release only at the lowest concentrations tested (10
−12
–10
−10
M). As the leptin concentration was increased, LHRH release decreased and was significantly less than control release at the highest concentration tested (10
−6
M). To determine if leptin can also release gonadotropins
in vivo
, ovariectomized females bearing implanted third ventricle cannulae were injected with 10 μg of estradiol benzoate s.c., followed 72 hr later by microinjection into the third ventricle of leptin (0.6 nmol in 5 μl) or an equal volume of diluent. There was a highly significant increase in plasma LH, which peaked 10–50 min after injection of leptin. Leptin had no effect on plasma FSH concentrations, and the diluent had no effect on either plasma FSH or LH. Thus, leptin at very low concentrations stimulated LHRH release from hypothalamic explants and FSH and LH release from anterior pituitaries of adult male rats
in vitro
and released LH, but not FSH,
in vivo
. The results indicate that leptin plays an important role in controlling gonadotropin secretion by stimulatory hypothalamic and pituitary actions.
Identification of an animal ω-3 fatty acid desaturase by heterologous expression in
Arabidopsis
Authors: Spychalla James P. , Kinney Anthony J. , Browse John
Published: July 2002

In animals, fatty acid desaturases catalyze key reactions in the synthesis of arachidonic acid and other polyunsaturated fatty acids. A search of the
Caenorhabditis elegans
DNA databases, using the sequences of
Arabidopsis
genes, identified several putative desaturases. Here we describe the characterization of the first of these genes,
fat-1
. The predicted protein encoded by a
fat-1
cDNA showed 32–35% identity with both FAD2 and FAD3 of
Arabidopsis
. When expressed in transgenic plants,
fat-1
resulted in a 90% increase in the proportion of α-linolenic acid in root lipids. Wild-type
Arabidopsis
incorporated ω-6 fatty acids (Δ8,11,14–20:3 and Δ5,8,11,14–20:4) into membrane lipids but did not desaturate them. By contrast,
fat-1
transgenic plants efficiently desaturated both of these fatty acids to the corresponding ω-3 products. These findings indicate that the
C. elegans fat-1
gene encodes the first animal representative of a class of glycerolipid desaturases that have previously been characterized in plants and cyanobacteria. The FAT-1 protein is an ω-3 fatty acyl desaturase that recognizes a range of 18- and 20-carbon ω-6 substrates.
Rapid reorganization of resistance gene homologues in cereal genomes
Authors: Leister Dario , Kurth Joachim , Laurie David A. , Yano Masahiro , Sasaki Takuji , Devos Katrien , Graner Andreas , Schulze-Lefert Paul
Published: July 2002

We used conserved domains in the major class (nucleotide binding site plus leucine-rich repeat) of dicot resistance (
R
) genes to isolate related gene fragments via PCR from the monocot species rice and barley. Peptide sequence comparison of dicot
R
genes and monocot
R
-like genes revealed shared motifs but provided no evidence for a monocot-specific signature. Mapping of these genes in rice and barley showed linkage to genetically characterized
R
genes and revealed the existence of mixed clusters, each harboring at least two highly dissimilar
R
-like genes. Diversity was detected intraspecifically with wide variation in copy number between varieties of a particular species. Interspecific analyses of
R
-like genes frequently revealed nonsyntenic map locations between the cereal species rice, barley, and foxtail millet although tight collinear gene order is a hallmark of monocot genomes. Our data suggest a dramatic rearrangement of
R
gene loci between related species and implies a different mechanism for nucleotide binding site plus leucine-rich repeat gene evolution compared with the rest of the monocot genome.
Generation of broad-spectrum disease resistance by overexpression of an essential regulatory gene in systemic acquired resistance
Authors: Cao Hui , Li Xin , Dong Xinnian
Published: July 2002

The recently cloned
NPR1
gene of
Arabidopsis thaliana
is a key regulator of acquired resistance responses. Upon induction,
NPR1
expression is elevated and the NPR1 protein is activated, in turn inducing expression of a battery of downstream pathogenesis-related genes. In this study, we found that NPR1 confers resistance to the pathogens
Pseudomonas syringae
and
Peronospora parasitica
in a dosage-dependent fashion. Overexpression of NPR1 leads to enhanced resistance with no obvious detrimental effect on the plants. Thus, for the first time, a single gene is shown to be a workable target for genetic engineering of nonspecific resistance in plants.
The complete genomic sequence of 424,015 bp at the centromeric end of the HLA class I region: Gene content and polymorphism
Authors: Guillaudeux Thierry , Janer Marta , Wong Gane Ka-Shu , Spies Thomas , Geraghty Daniel E.
Published: July 2002

We report here the genomic sequence of the centromeric portion of HLA class I, extending 424,015 bp from tumor necrosis factor α to a newly identified gene ≈20 kb telomeric of Otf-3. As a source of DNA, we used cosmids centromeric of HLA-B that had been mapped previously with conventional restriction digestion and fingerprinting and previously characterized yeast artificial chromosomes subcloned into cosmids and mapped with multiple complete digest methodologies. The data presented provide a description of the gene content of centromeric HLA class I including new data on intron, promoter and flanking sequences of previously described genes, and a description of putative new genes that remain to be characterized beyond the structural information uncovered. A complete accounting of the repeat structure including abundant di-, tri-, and tetranucleotide microsatellite loci yielded access to precisely localized mapping tools for the major histocompatibility complex. Comparative analysis of a highly polymorphic region between HLA-B and -C was carried out by sequencing over 40 kb of overlapping sequence from two haplotypes. The levels of variation observed were much higher than those seen in other regions of the genome and indeed were higher than those observed between allelic HLA class I loci.
Evidence for balancing selection operating at the
het
-c heterokaryon incompatibility locus in a group of filamentous fungi
Authors: Wu Jennifer , Saupe Sven J. , Glass N. Louise
Published: July 2002

In filamentous fungi,
het
loci (for
het
erokaryon incompatibility) are believed to regulate self/nonself-recognition during vegetative growth. As filamentous fungi grow, hyphal fusion occurs within an individual colony to form a network. Hyphal fusion can occur also between different individuals to form a heterokaryon, in which genetically distinct nuclei occupy a common cytoplasm. However, heterokaryotic cells are viable only if the individuals involved have identical alleles at all
het
loci. One
het
locus,
het-c
, has been characterized at the molecular level in
Neurospora crassa
and encodes a glycine-rich protein. In an effort to understand the role of this locus in filamentous fungi, we chose to study its evolution by analyzing
het-c
sequence variability in species within
Neurospora
and related genera. We determined that the
het-c
locus was polymorphic in a field population of
N. crassa
with close to equal frequency of each of the three allelic types. Different species and even genera within the Sordariaceae shared
het-c
polymorphisms, indicating that these polymorphisms originated in an ancestral species. Finally, an analysis of the
het-c
specificity region shows a high occurrence of nonsynonymous substitution. The persistence of allelic lineages, the nearly equal allelic distribution within populations, and the high frequency of nonsynonymous substitutions in the
het-c
specificity region suggest that balancing selection has operated to maintain allelic diversity at
het-c. Het-c
shares this particular evolutionary characteristic of departing from neutrality with other self/nonself-recognition systems such as major histocompatibility complex loci in mammals and the
S
(self-incompatibility) locus in angiosperms.
Identification and analysis of the plant peroxisomal targeting signal 1 receptor NtPEX5
Authors: Kragler Friedrich , Lametschwandtner Günther , Christmann James , Hartig Andreas , Harada John J.
Published: July 2002

Protein translocation into peroxisomes takes place via recognition of a peroxisomal targeting signal present at either the extreme C termini (PTS1) or N termini (PTS2) of matrix proteins. In mammals and yeast, the peroxisomal targeting signal receptor, Pex5p, recognizes the PTS1 consisting of -SKL or variants thereof. Although many plant peroxisomal matrix proteins are transported through the PTS1 pathway, little is known about the PTS1 receptor or any other peroxisome assembly protein from plants. We cloned tobacco (
Nicotiana tabacum
) cDNAs encoding Pex5p (NtPEX5) based on the protein’s interaction with a PTS1-containing protein in the yeast two-hybrid system. Nucleotide sequence analysis revealed that the tobacco Pex5p contains seven tetratricopeptide repeats and that NtPEX5 shares greater sequence similarity with its homolog from humans than from yeast. Expression of NtPEX5 fusion proteins, consisting of the N-terminal part of yeast Pex5p and the C-terminal region of NtPEX5, in a
Saccharomyces cerevisiae pex5
mutant restored protein translocation into peroxisomes. These experiments confirmed the identity of the tobacco protein as a PTS1 receptor and indicated that components of the peroxisomal translocation apparatus are conserved functionally. Two-hybrid assays showed that NtPEX5 interacts with a wide range of PTS1 variants that also interact with the human Pex5p. Interestingly, the C-terminal residues of some of these peptides deviated from the established plant PTS1 consensus sequence. We conclude that there are significant sequence and functional similarities between the plant and human Pex5ps.
Molecular cloning and expression of the human Δ7-sterol reductase
Authors: Moebius Fabian F. , Fitzky Barbara U. , Lee Joon No , Paik Young-Ki , Glossmann Hartmut
Published: July 2002

Inhibitors of the last steps of cholesterol biosynthesis such as AY9944 and BM15766 severely impair brain development. Their molecular target is the Δ7-sterol reductase (EC
1.3.1.21
), suspected to be defective in the Smith–Lemli–Opitz syndrome, a frequent inborn disorder of sterol metabolism. Molecular cloning of the cDNA revealed that the human enzyme is a membrane-bound protein with a predicted molecular mass of 55 kDa and six to nine putative transmembrane segments. The protein is structurally related to plant and yeast sterol reductases. In adults the ubiquitously transcribed mRNA is most abundant in adrenal gland, liver, testis, and brain. The Δ7-sterol reductase is the ultimate enzyme of cholesterol biosynthesis in vertebrates and is absent from yeast. Microsomes from
Saccharomyces cerevisiae
strains heterologously expressing the human cDNA remove the C
7–8
double bond in 7-dehydrocholesterol. The conversion to cholesterol depends on NADPH and is potently inhibited by AY9944 (IC
50
0.013 μM), BM15766 (IC
50
1.2 μM), and triparanol (IC
50
14 μM). Our work paves the way to clarify whether a defect in the Δ7-sterol reductase gene underlies the Smith–Lemli–Opitz syndrome.
Isolation of a
Drosophila
homolog of the vertebrate homeobox gene Rx and its possible role in brain and eye development
Authors: Eggert Tanja , Hauck Bernd , Hildebrandt Nicole , Gehring Walter J. , Walldorf Uwe
Published: July 2002

Vertebrate and invertebrate eye development require the activity of several evolutionarily conserved genes. Among these the
Pax-6
genes play a major role in the genetic control of eye development. Mutations in
Pax-6
genes affect eye development in humans, mice, and
Drosophila,
and misexpression of
Pax-6
genes in
Drosophila
can induce ectopic eyes. Here we report the identification of a paired-like homeobox gene, DRx, which is also conserved from flies to vertebrates. Highly conserved domains in the
Drosophila
protein are the octapeptide, the identical homeodomain, the carboxyl-terminal OAR domain, and a newly identified Rx domain. DRx is expressed in the embryo in the procephalic region and in the clypeolabrum from stage 8 on and later in the brain and the central nervous system. Compared with
eyeless,
the DRx expression in the embryo starts earlier, similar to the pattern in vertebrates, where Rx expression precedes
Pax-6
expression. Because the vertebrate Rx genes have a function during brain and eye development, it was proposed that DRx has a similar function. The DRx expression pattern argues for a conserved function at least during brain development, but we could not detect any expression in the embryonic eye primordia or in the larval eye imaginal discs. Therefore DRx could be considered as a homolog of vertebrate Rx genes. The Rx genes might be involved in brain patterning processes and specify eye fields in different phyla.
Structure and mechanism of a proline-specific aminopeptidase from
Escherichia coli
Authors: Wilce M. C. J. , Bond C. S. , Dixon N. E. , Freeman H. C. , Guss J. M. , Lilley P. E. , Wilce J. A.
Published: July 2002

The structure of the proline-specific aminopeptidase (EC
3.4.11.9
) from
Escherichia coli
has been solved and refined for crystals of the native enzyme at a 2.0-Å resolution, for a dipeptide-inhibited complex at 2.3-Å resolution, and for a low-pH inactive form at 2.7-Å resolution. The protein crystallizes as a tetramer, more correctly a dimer of dimers, at both high and low pH, consistent with observations from analytical ultracentrifuge studies that show that the protein is a tetramer under physiological conditions. The monomer folds into two domains. The active site, in the larger C-terminal domain, contains a dinuclear manganese center in which a bridging water molecule or hydroxide ion appears poised to act as the nucleophile in the attack on the scissile peptide bond of Xaa-Pro. The metal-binding residues are located in a single subunit, but the residues surrounding the active site are contributed by three subunits. The fold of the protein resembles that of creatine amidinohydrolase (creatinase, not a metalloenzyme). The C-terminal catalytic domain is also similar to the single-domain enzyme methionine aminopeptidase that has a dinuclear cobalt center.
Disordered water within a hydrophobic protein cavity visualized by x-ray crystallography
Authors: Yu B. , Blaber M. , Gronenborn A. M. , Clore G. M. , Caspar D. L. D.
Published: July 2002

Water in the hydrophobic cavity of human interleukin 1β, which was detected by NMR spectroscopy but was invisible by high resolution x-ray crystallography, has been mapped quantitatively by measurement and phasing of all of the low resolution x-ray diffraction data from a single crystal. Phases for the low resolution data were refined by iterative density modification of an initial flat solvent model outside the envelope of the atomic model. The refinement was restrained by the condition that the map of the difference between the electron density distribution in the full unit cell and that of the atomic model be flat within the envelope of the well ordered protein structure. Care was taken to avoid overfitting the diffraction data by maintaining phases for the high resolution data from the atomic model and by a resolution-dependent damping of the structure factor differences between data and model. The cavity region in the protein could accommodate up to four water molecules. The refined solvent difference map indicates that there are about two water molecules in the cavity region. This map is compatible with an atomic model of the water distribution refined by using
xplor
. About 70% of the time, there appears to be a water dimer in the central hydrophobic cavity, which is connected to the outside by two constricted channels occupied by single water molecules ≈40% of the time on one side and ≈10% on the other.
Clonal selection and
in vivo
quantitation of protein interactions with protein-fragment complementation assays
Authors: Remy Ingrid , Michnick Stephen W.
Published: July 2002

Two strategies are described for detecting constitutive or induced protein–protein interactions in intact mammalian cells; these strategies are based on oligomerization domain-assisted complementation of rationally designed fragments of the murine enzyme dihydrofolate reductase (DHFR; EC
1.5.1.3
). We describe a dominant clonal-selection assay of stably transfected cells expressing partner proteins FKBP (FK506 binding protein) and FRAP (FKBP–rapamycin binding protein) fused to DHFR fragments and show a rapamycin dose-dependent survival of clones that requires ≈25 molecules of reconstituted DHFR per cell. A fluorescence assay also is described, based on stoichiometric binding of fluorescein-methotrexate to reconstituted DHFR
in vivo
. Formation of the FKBP–rapamycin–FRAP complex is detected in stably and transiently transfected cells. Quantitative rapamycin dose-dependence of this complex is shown to be consistent with
in vitro
binding and distinguishable from a known constitutive interaction of FKBP and FRAP. We also show that this strategy can be applied to study membrane protein receptors, demonstrating dose-dependent activation of the erythropoietin receptor by ligands. The combination of these clonal-selection and fluorescence assays in intact mammalian cells makes possible selection by simple survival, flow cytometry, or both. High-throughput drug screening and quantitative analysis of induction or disruption of protein–protein interactions are also made possible.
Intranuclear localization of human papillomavirus 16 E7 during transformation and preferential binding of E7 to the Rb family member p130
Authors: Smith-McCune K. , Kalman D. , Robbins C. , Shivakumar S. , Yuschenkoff L. , Bishop J. Michael
Published: July 2002

To study intracellular pathways by which the human papillomavirus 16 oncogene E7 participates in carcinogenesis, we expressed an inducible chimera of E7 by fusion to the hormone-binding domain of the estrogen receptor. The chimeric protein (E7ER) transformed rodent fibroblast cell lines and induced DNA synthesis on addition of estradiol. In coimmunoprecipitation experiments, E7ER preferentially bound p130 when compared to p107 and pRb. After estradiol addition, E7ER localization changed to a more intense intranuclear staining. Induction of E7 function was not correlated with binding to p130 or pRb but rather with intranuclear localization and modest induction of binding to p107.
Map-based cloning of
chloronerva,
a gene involved in iron uptake of higher plants encoding nicotianamine synthase
Authors: Ling Hong-Qing , Koch Gudrun , Bäumlein Helmut , Ganal Martin W.
Published: July 2002

The uptake of iron in plants is a highly regulated process that is induced on iron starvation. In tomato, the mutant
chloronerva
exhibits constitutive expression of iron uptake responses and intercostal chlorosis. Biochemically,
chloronerva
is an auxotroph for nicotianamine, a key polyamine in plant iron uptake metabolism. The
chloronerva
gene has been fine-mapped onto the long arm of chromosome 1 in a large segregating tomato population and yeast artificial chromosome clones encompassing the region were isolated by using flanking markers. A cosmid contig containing the
chloronerva
gene was established, and complementing cosmids were identified by transformation into the mutant. The
chloronerva
transcript was identified by cDNA isolation using the complementing cosmids. The gene encodes a unique protein of 35 kDa. The mutant harbors a single base change compared with the wild type. Based on enzyme activity and sequence similarity to the coding DNA sequence of the purified barley enzyme the
chloronerva
gene encodes the enzyme nicotianamine synthase.
Protective immunity against murine hepatitis virus (MHV) induced by intranasal or subcutaneous administration of hybrids of tobacco mosaic virus that carries an MHV epitope
Authors: Koo Moses , Bendahmane Mohammed , Lettieri Gerard A. , Paoletti Alyssa D. , Lane Thomas E. , Fitchen John H. , Buchmeier Michael J. , Beachy Roger N.
Published: July 2002

Hybrids of tobacco mosaic virus (TMV) were constructed with the use of fusion to the coat protein peptides of 10 or 15 amino acids, containing the 5B19 epitope from the spike protein of murine hepatitis virus (MHV) and giving rise to TMV-5B19 and TMV-5B19L, respectively. The TMV hybrids were propagated in tobacco plants, and the virus particles were purified. Immunogold labeling, with the use of the monoclonal MAb5B19 antibody, showed specific decoration of hybrid TMV particles, confirming the expression and display of the MHV epitope on the surface of the TMV. Mice were immunized with purified hybrid viruses after several regimens of immunization. Mice that received TMV-5B19L intranasally developed serum IgG and IgA specific for the 5B19 epitope and for the TMV coat protein. Hybrid TMV-5B19, administered by subcutaneous injections, elicited high titers of serum IgG that was specific for the 5B19 epitope and for coat protein, but IgA that was specific against 5B19 was not observed. Mice that were immunized with hybrid virus by subcutaneous or intranasal routes of administration survived challenge with a lethal dose (10 × LD
50
) of MHV strain JHM, whereas mice administered wild-type TMV died 10 d post challenge. Furthermore, there was a positive correlation between the dose of administered immunogen and protection against MHV infection. These studies show that TMV can be an effective vaccine delivery vehicle for parenteral and mucosal immunization and for protection from challenge with viral infection.
Crystallographic analysis of CD40 recognition and signaling by human TRAF2
Authors: McWhirter Sarah M. , Pullen Steven S. , Holton James M. , Crute James J. , Kehry Marilyn R. , Alber Tom
Published: July 2002

Tumor necrosis factor receptor superfamily members convey signals that promote diverse cellular responses. Receptor trimerization by extracellular ligands initiates signaling by recruiting members of the tumor necrosis factor receptor-associated factor (TRAF) family of adapter proteins to the receptor cytoplasmic domains. We report the 2.4-Å crystal structure of a 22-kDa, receptor-binding fragment of TRAF2 complexed with a functionally defined peptide from the cytoplasmic domain of the CD40 receptor. TRAF2 forms a mushroom-shaped trimer consisting of a coiled coil and a unique β-sandwich domain. Both domains mediate trimerization. The CD40 peptide binds in an extended conformation with every side chain in contact with a complementary groove on the rim of each TRAF monomer. The spacing between the CD40 binding sites on TRAF2 supports an elegant signaling mechanism in which trimeric, extracellular ligands preorganize the receptors to simultaneously recognize three sites on the TRAF trimer.