bluuebunny/crossref_metadata_2025 huggingface dataset:
Universally conserved translation initiation factors
Authors: Kyrpides Nikos C. , Woese Carl R.
Published: July 2002

The process by which translation is initiated has long been considered similar in Bacteria and Eukarya but accomplished by a different unrelated set of factors in the two cases. This not only implies separate evolutionary histories for the two but also implies that at the universal ancestor stage, a translation initiation mechanism either did not exist or was of a different nature than the extant processes. We demonstrate herein that (
i
) the “analogous” translation initiation factors IF-1 and eIF-1A are actually related in sequence, (
ii
) the “eukaryotic” translation factor SUI1 is universal in distribution, and (
iii
) the eukaryotic/archaeal translation factor eIF-5A is homologous to the bacterial translation factor EF-P. Thus, the rudiments of translation initiation would seem to have been present in the universal ancestor stage. However, significant development and refinement subsequently occurred independently on both the bacterial lineage and on the archaeal/eukaryotic line.
Natural killer-like nonspecific tumor cell lysis mediated by specific ligand-activated Vα14 NKT cells
Authors: Kawano Tetsu , Cui Junqing , Koezuka Yasuhiko , Toura Isao , Kaneko Yoshikatsu , Sato Hiroshi , Kondo Eisuke , Harada Michishige , Koseki Haruhiko , Nakayama Toshinori , Tanaka Yujiro , Taniguchi Masaru
Published: July 2002

We have recently identified α-galactosylceramide (α-GalCer) as a specific ligand for an invariant Vα14/Vβ8.2 T cell receptor exclusively expressed on the majority of Vα14 NKT cells, a novel subset of lymphocytes. Here, we report that α-GalCer selectively activates Vα14 NKT cells resulting in prevention of tumor metastasis. The effector mechanisms of the ligand-activated Vα14 NKT cells seem to be mediated by natural killer (NK)-like nonspecific cytotoxicity. Indeed, the cytotoxic index obtained by α-GalCer-activated Vα14 NKT cells was reduced by the addition of cold target tumor cells or by treatment with concanamycin A, which inhibits activation and secretion of perforin, but not by mAbs against molecules involved in the NKT cell recognition and conventional cytotoxicity, such as CD1d, Vβ8, NK1.1, Ly49C, Fas, or Fas ligand. These results suggest that the ligand-activated Vα14 NKT cells kill tumor cells directly through a CD1d/Vα14 T cell receptor-independent, NK-like mechanism.
An amino acid as a cofactor for a catalytic polynucleotide
Authors: Roth Adam , Breaker Ronald R.
Published: July 2002

Natural ribozymes require metal ion cofactors that aid both in structural folding and in chemical catalysis. In contrast, many protein enzymes produce dramatic rate enhancements using only the chemical groups that are supplied by their constituent amino acids. This fact is widely viewed as the most important feature that makes protein a superior polymer for the construction of biological catalysts. Herein we report the
in vitro
selection of a catalytic DNA that uses histidine as an active component for an RNA cleavage reaction. An optimized deoxyribozyme from this selection requires
l
-histidine or a closely related analog to catalyze RNA phosphoester cleavage, producing a rate enhancement of ≈1-million-fold over the rate of substrate cleavage in the absence of enzyme. Kinetic analysis indicates that a DNA–histidine complex may perform a reaction that is analogous to the first step of the proposed catalytic mechanism of RNase A, in which the imidazole group of histidine serves as a general base catalyst. Similarly, ribozymes of the “RNA world” may have used amino acids and other small organic cofactors to expand their otherwise limited catalytic potential.
Diffusible, nonfibrillar ligands derived from Aβ
1–42
are potent central nervous system neurotoxins
Authors: Lambert M. P. , Barlow A. K. , Chromy B. A. , Edwards C. , Freed R. , Liosatos M. , Morgan T. E. , Rozovsky I. , Trommer B. , Viola K. L. , Wals P. , Zhang C. , Finch C. E. , Krafft G. A. , Klein W. L.
Published: July 2002

Aβ
1–42
is a self-associating peptide whose neurotoxic derivatives are thought to play a role in Alzheimer’s pathogenesis. Neurotoxicity of amyloid β protein (Aβ) has been attributed to its fibrillar forms, but experiments presented here characterize neurotoxins that assemble when fibril formation is inhibited. These neurotoxins comprise small diffusible Aβ oligomers (referred to as ADDLs, for Aβ-derived diffusible ligands), which were found to kill mature neurons in organotypic central nervous system cultures at nanomolar concentrations. At cell surfaces, ADDLs bound to trypsin-sensitive sites and surface-derived tryptic peptides blocked binding and afforded neuroprotection. Germ-line knockout of Fyn, a protein tyrosine kinase linked to apoptosis and elevated in Alzheimer’s disease, also was neuroprotective. Remarkably, neurological dysfunction evoked by ADDLs occurred well in advance of cellular degeneration. Without lag, and despite retention of evoked action potentials, ADDLs inhibited hippocampal long-term potentiation, indicating an immediate impact on signal transduction. We hypothesize that impaired synaptic plasticity and associated memory dysfunction during early stage Alzheimer’s disease and severe cellular degeneration and dementia during end stage could be caused by the biphasic impact of Aβ-derived diffusible ligands acting upon particular neural signal transduction pathways.
Measurement of cytosolic, mitochondrial, and Golgi pH in single living cells with green fluorescent proteins
Authors: Llopis Juan , McCaffery J. Michael , Miyawaki Atsushi , Farquhar Marilyn G. , Tsien Roger Y.
Published: July 2002

Many cellular events depend on a tightly compartmentalized distribution of H
+
ions across membrane-bound organelles. However, measurements of organelle pH in living cells have been scarce. Several mutants of the
Aequorea victoria
green fluorescent protein (GFP) displayed a pH-dependent absorbance and fluorescent emission, with apparent pKa values ranging from 6.15 (mutations F64L/S65T/H231L) and 6.4 (K26R/F64L/S65T/Y66W/N146I/M153T/V163A/N164H/H231L) to a remarkable 7.1 (S65G/S72A/T203Y/H231L). We have targeted these GFPs to the cytosol plus nucleus, the medial/trans-Golgi by fusion with galactosyltransferase, and the mitochondrial matrix by using the targeting signal from subunit IV of cytochrome
c
oxidase. Cells in culture transfected with these cDNAs displayed the expected subcellular localization by light and electron microscopy and reported local pH that was calibrated
in situ
with ionophores. We monitored cytosolic and nuclear pH of HeLa cells, and mitochondrial matrix pH in HeLa cells and in rat neonatal cardiomyocytes. The pH of the medial/trans-Golgi was measured at steady-state (calibrated to be 6.58 in HeLa cells) and after various manipulations. These demonstrated that the Golgi membrane in intact cells is relatively permeable to H
+
, and that Cl
−
serves as a counter-ion for H
+
transport and likely helps to maintain electroneutrality. The amenability to engineer GFPs to specific subcellular locations or tissue targets using gene fusion and transfer techniques should allow us to examine pH at sites previously inaccessible.
Incidence and functional consequences of
hMLH1
promoter hypermethylation in colorectal carcinoma
Authors: Herman James G. , Umar Asad , Polyak Kornelia , Graff Jeremy R. , Ahuja Nita , Issa Jean-Pierre J. , Markowitz Sanford , Willson James K. V. , Hamilton Stanley R. , Kinzler Kenneth W. , Kane Michael F. , Kolodner Richard D. , Vogelstein Bert , Kunkel Thomas A. , Baylin Stephen B.
Published: July 2002

Inactivation of the genes involved in DNA mismatch repair is associated with microsatellite instability (MSI) in colorectal cancer. We report that hypermethylation of the 5′ CpG island of
hMLH1
is found in the majority of sporadic primary colorectal cancers with MSI, and that this methylation was often, but not invariably, associated with loss of hMLH1 protein expression. Such methylation also occurred, but was less common, in MSI− tumors, as well as in MSI+ tumors with known mutations of a mismatch repair gene (MMR). No hypermethylation of
hMSH2
was found. Hypermethylation of colorectal cancer cell lines with MSI also was frequently observed, and in such cases, reversal of the methylation with 5-aza-2′-deoxycytidine not only resulted in reexpression of hMLH1 protein, but also in restoration of the MMR capacity in MMR-deficient cell lines. Our results suggest that microsatellite instability in sporadic colorectal cancer often results from epigenetic inactivation of
hMLH1
in association with DNA methylation.
Regulation of hypoxia-inducible factor 1α is mediated by an O
2
-dependent degradation domain via the ubiquitin-proteasome pathway
Authors: Huang L. Eric , Gu Jie , Schau Maureen , Bunn H. Franklin
Published: July 2002

Hypoxia induces a group of physiologically important genes such as erythropoietin and vascular endothelial growth factor. These genes are transcriptionally up-regulated by hypoxia-inducible factor 1 (HIF-1), a global regulator that belongs to the basic helix-loop-helix PAS family. Although HIF-1 is a heterodimer composed of α and β subunits, its activity is primarily determined by hypoxia-induced stabilization of HIF-1α, which is otherwise rapidly degraded in oxygenated cells. We report the identification of an oxygen-dependent degradation (ODD) domain within HIF-1α that controls its degradation by the ubiquitin-proteasome pathway. The ODD domain consists of ≈200 amino acid residues, located in the central region of HIF-1α. Because portions of the domain independently confer degradation of HIF-1α, deletion of this entire region is required to give rise to a stable HIF-1α, capable of heterodimerization, DNA-binding, and transactivation in the absence of hypoxic signaling. Conversely, the ODD domain alone confers oxygen-dependent instability when fused to a stable protein, Gal4. Hence, the ODD domain plays a pivotal role for regulating HIF-1 activity and thereby may provide a means of controlling gene expression by changes in oxygen tension.
Racial variability in the UDP-glucuronosyltransferase 1 (
UGT1A1
) promoter: A balanced polymorphism for regulation of bilirubin metabolism?
Authors: Beutler Ernest , Gelbart Terri , Demina Anna
Published: July 2002

A polymorphism in the promoter of the UDP-glucuronosyltransferase 1 (
UGT1A1
) gene has been shown to cause Gilbert syndrome, a benign form of unconjugated bilirubinemia. Promoters containing seven thymine adenine (ta) repeats have been found to be less active than the wild-type six repeats, and the serum bilirubin levels of persons homozygous or even heterozygous for seven repeats have been found to be higher than those with the wild-type six repeats. We have now examined the genotypes in persons of Asian, African, and Caucasian ancestry. Although within the Caucasian ethnic group there is a strong correlation between promoter repeat number and bilirubin level, between ethnic groups we found that this relationship to be inverse. Among people of African ancestry there are, in addition to those with six and seven repeats, also persons who have five or eight repeats. Using a reporter gene we show that there is an inverse relationship between the number of ta repeats and the activity of the promoter through the range of 5–8 ta repeats. An incidental finding was a polymorphism at nucleotide −106, tightly linked to the (ta)
5
haplotype. Serum bilirubin levels are influenced by many factors, both genetic and environmental. We suggest that the unstable
UGT1A1
polymorphism may serve to “fine-tune” the plasma bilirubin level within population groups, maintaining it at a high enough level to provide protection against oxidative damage, but at a level that is sufficiently low to prevent kernicterus in infants.
Molecular archaeology of the
Escherichia coli
genome
Authors: Lawrence Jeffrey G. , Ochman Howard
Published: July 2002

The availability of the complete sequence of
Escherichia coli
strain MG1655 provides the first opportunity to assess the overall impact of horizontal genetic transfer on the evolution of bacterial genomes. We found that 755 of 4,288 ORFs (547.8 kb) have been introduced into the
E. coli
genome in at least 234 lateral transfer events since this species diverged from the
Salmonella
lineage 100 million years (Myr) ago. The average age of introduced genes was 14.4 Myr, yielding a rate of transfer 16 kb/Myr/lineage since divergence. Although most of the acquired genes subsequently were deleted, the sequences that have persisted (≈18% of the current chromosome) have conferred properties permitting
E. coli
to explore otherwise unreachable ecological niches.
Selective effects of methylphenidate in attention deficit hyperactivity disorder: A functional magnetic resonance study
Authors: Vaidya Chandan J. , Austin Glenn , Kirkorian Gary , Ridlehuber Hugh W. , Desmond John E. , Glover Gary H. , Gabrieli John D. E.
Published: July 2002

Functional MRI revealed differences between children with Attention Deficit Hyperactivity Disorder (ADHD) and healthy controls in their frontal–striatal function and its modulation by methylphenidate during response inhibition. Children performed two go/no-go tasks with and without drug. ADHD children had impaired inhibitory control on both tasks. Off-drug frontal–striatal activation during response inhibition differed between ADHD and healthy children: ADHD children had greater frontal activation on one task and reduced striatal activation on the other task. Drug effects differed between ADHD and healthy children: The drug improved response inhibition in both groups on one task and only in ADHD children on the other task. The drug modulated brain activation during response inhibition on only one task: It increased frontal activation to an equal extent in both groups. In contrast, it increased striatal activation in ADHD children but reduced it in healthy children. These results suggest that ADHD is characterized by atypical frontal–striatal function and that methylphenidate affects striatal activation differently in ADHD than in healthy children.
Semirational design of active tumor suppressor p53 DNA binding domain with enhanced stability
Authors: Nikolova Penka V. , Henckel Julia , Lane David P. , Fersht Alan R.
Published: July 2002

We have designed a p53 DNA binding domain that has virtually the same binding affinity for the
gadd45
promoter as does wild-type protein but is considerably more stable. The design strategy was based on molecular evolution of the protein domain. Naturally occurring amino acid substitutions were identified by comparing the sequences of p53 homologues from 23 species, introducing them into wild-type human p53, and measuring the changes in stability. The most stable substitutions were combined in a multiple mutant. The advantage of this strategy is that, by substituting with naturally occurring residues, the function is likely to be unimpaired. All point mutants bind the consensus DNA sequence. The changes in stability ranged from +1.27 (less stable Q165K) to −1.49 (more stable N239Y) kcal mol
−1
, respectively. The changes in free energy of unfolding on mutation are additive. Of interest, the two most stable mutants (N239Y and N268D) have been known to act as suppressors and restored the activity of two of the most common tumorigenic mutants. Of the 20 single mutants, 10 are cancer-associated, though their frequency of occurrence is extremely low: A129D, Q165K, Q167E, and D148E are less stable and M133L, V203A and N239Y are more stable whereas the rest are neutral. The quadruple mutant (M133LV203AN239YN268D), which is stabilized by 2.65 kcal mol
−1
and
T
m
raised by 5.6°C is of potential interest for trials
in vivo
.
A model for amplification of hair-bundle motion by cyclical binding of Ca
2+
to mechanoelectrical-transduction channels
Authors: Choe Yong , Magnasco Marcelo O. , Hudspeth A. J.
Published: July 2002

Amplification of auditory stimuli by hair cells augments the sensitivity of the vertebrate inner ear. Cell-body contractions of outer hair cells are thought to mediate amplification in the mammalian cochlea. In vertebrates that lack these cells, and perhaps in mammals as well, active movements of hair bundles may underlie amplification. We have evaluated a mathematical model in which amplification stems from the activity of mechanoelectrical-transduction channels. The intracellular binding of Ca
2+
to channels is posited to promote their closure, which increases the tension in gating springs and exerts a negative force on the hair bundle. By enhancing bundle motion, this force partially compensates for viscous damping by cochlear fluids. Linear stability analysis of a six-state kinetic model reveals Hopf bifurcations for parameter values in the physiological range. These bifurcations signal conditions under which the system’s behavior changes from a damped oscillatory response to spontaneous limit-cycle oscillation. By varying the number of stereocilia in a bundle and the rate constant for Ca
2+
binding, we calculate bifurcation frequencies spanning the observed range of auditory sensitivity for a representative receptor organ, the chicken’s cochlea. Simulations using prebifurcation parameter values demonstrate frequency-selective amplification with a striking compressive nonlinearity. Because transduction channels occur universally in hair cells, this active-channel model describes a mechanism of auditory amplification potentially applicable across species and hair-cell types.
Modulation of
N
-methyl-
d
-aspartate receptor function by glycine transport
Authors: Bergeron Richard , Meyer Torsten M. , Coyle Joseph T. , Greene Robert W.
Published: July 2002

The recent discovery of glycine transporters in both the central nervous system and the periphery suggests that glycine transport may be critical to
N
-methyl-
d
-aspartate receptor (NMDAR) function by controlling glycine concentration at the NMDAR modulatory glycine site. Data obtained from whole-cell patch–clamp recordings of hippocampal pyramidal neurons,
in vitro
, demonstrated that exogenous glycine and glycine transporter type 1 (GLYT1) antagonist selectively enhanced the amplitude of the NMDA component of a glutamatergic excitatory postsynaptic current. The effect was blocked by 2-amino-5-phosphonovaleric acid and 7-chloro-kynurenic acid but not by strychnine. Thus, the glycine-binding site was not saturated under the control conditions. Furthermore, GLYT1 antagonist enhanced NMDAR function during perfusion with medium containing 10 μM glycine, a concentration similar to that in the cerebrospinal fluid
in vivo
, thereby supporting the hypothesis that the GLYT1 maintains subsaturating concentration of glycine at synaptically activated NMDAR. The enhancement of NMDAR function by specific GLYT1 antagonism may be a feasible target for therapeutic agents directed toward diseases related to hypofunction of NMDAR.
Event-related brain potentials in the study of visual selective attention
Authors: Hillyard Steven A. , Anllo-Vento Lourdes
Published: July 2002

Event-related brain potentials (ERPs) provide high-resolution measures of the time course of neuronal activity patterns associated with perceptual and cognitive processes. New techniques for ERP source analysis and comparisons with data from blood-flow neuroimaging studies enable improved localization of cortical activity during visual selective attention. ERP modulations during spatial attention point toward a mechanism of gain control over information flow in extrastriate visual cortical pathways, starting about 80 ms after stimulus onset. Paying attention to nonspatial features such as color, motion, or shape is manifested by qualitatively different ERP patterns in multiple cortical areas that begin with latencies of 100–150 ms. The processing of nonspatial features seems to be contingent upon the prior selection of location, consistent with early selection theories of attention and with the hypothesis that spatial attention is “special.”
New protein kinase and protein phosphatase families mediate signal transduction in bacterial catabolite repression
Authors: Galinier Anne , Kravanja Melanie , Engelmann Roswitha , Hengstenberg Wolfgang , Kilhoffer Marie-Claude , Deutscher Josef , Haiech Jacques
Published: July 2002

Carbon catabolite repression (CCR) is the prototype of a signal transduction mechanism. In enteric bacteria, cAMP was considered to be the second messenger in CCR by playing a role reminiscent of its actions in eukaryotic cells. However, recent results suggest that CCR in
Escherichia coli
is mediated mainly by an inducer exclusion mechanism. In many Gram-positive bacteria, CCR is triggered by fructose-1,6-bisphosphate, which activates HPr kinase, presumed to be one of the most ancient serine protein kinases. We here report cloning of the
Bacillus subtilis hprK
and
hprP
genes and characterization of the encoded HPr kinase and P-Ser-HPr phosphatase. P-Ser-HPr phosphatase forms a new family of phosphatases together with bacterial phosphoglycolate phosphatase, yeast glycerol-3-phosphatase, and 2-deoxyglucose-6-phosphate phosphatase whereas HPr kinase represents a new family of protein kinases on its own. It does not contain the domain structure typical for eukaryotic protein kinases. Although up to now the HPr modifying/demodifying enzymes were thought to exist only in Gram-positive bacteria, a sequence comparison revealed that they also are present in several Gram-negative pathogenic bacteria.
Natriuretic peptide receptor 1 expression influences blood pressures of mice in a dose-dependent manner
Authors: Oliver Paula M. , John Simon W. M. , Purdy Kit E. , Kim Ron , Maeda Nobuyo , Goy Michael F. , Smithies Oliver
Published: July 2002

Activation of the natriuretic peptide system lowers blood pressure and causes the excretion of salt. Atrial natriuretic peptide and B-type natriuretic peptide are the humoral mediators of this effect; they act primarily by binding to membrane-bound natriuretic peptide receptor A (NPRA) and stimulating its intrinsic guanylate cyclase activity. To study whether genetically determined differences in NPRA expression affect blood pressure we have generated mice with one, two, three, or four copies of the gene encoding NPRA (
Npr1
in the mouse). Atrial natriuretic peptide-dependent guanylate cyclase activity ranged progressively from approximately one-half normal in one-copy animals to twice normal in four-copy animals (
P
< 0.001). On different diets (0.05%, 2%, and 8% NaCl), the blood pressures of F
1
male mice having only one copy of
Npr1
averaged 9.1 mmHg (1 mmHg = 133 Pa) above those of wild-type two-copy males (
P
< 0.001), whereas males with three copies of the gene had blood pressures averaging 5.2 mmHg below normal (
P
< 0.01). The blood pressures of the one-copy F
1
animals were significantly higher (by 6.2 mmHg;
P
< 0.01) on the high-salt than on the low-salt diet. The blood pressures of four-copy F
3
males were significantly lower (by 7 mmHg;
P
< 0.05) on the high-salt than on the low-salt diet. These results demonstrate that below normal
Npr1
expression leads to a salt-sensitive increase in blood pressure, whereas above normal
Npr1
expression lowers blood pressures and protects against high dietary salt.
Role of substance P and the neurokinin 1 receptor in acute pancreatitis and pancreatitis-associated lung injury
Authors: Bhatia Madhav , Saluja Ashok K. , Hofbauer Bernd , Frossard Jean-Louis , Lee Hong Sik , Castagliuolo Ignazio , Wang Chi-Chung , Gerard Norma , Pothoulakis Charalabos , Steer Michael L.
Published: July 2002

Substance P, acting via the neurokinin 1 receptor (NK1R), plays an important role in mediating a variety of inflammatory processes. However, its role in acute pancreatitis has not been previously described. We have found that, in normal mice, substance P levels in the pancreas and pancreatic acinar cell expression of NK1R are both increased during secretagogue-induced experimental pancreatitis. To evaluate the role of substance P, pancreatitis was induced in mice that genetically lack NK1R by administration of 12 hourly injections of a supramaximally stimulating dose of the secretagogue caerulein. During pancreatitis, the magnitude of hyperamylasemia, hyperlipasemia, neutrophil sequestration in the pancreas, and pancreatic acinar cell necrosis were significantly reduced in NK1R−/− mice when compared with wild-type NK1R+/+ animals. Similarly, pancreatitis-associated lung injury, as characterized by intrapulmonary sequestration of neutrophils and increased pulmonary microvascular permeability, was reduced in NK1R−/− animals. These effects of NK1R deletion indicate that substance P, acting via NK1R, plays an important proinflammatory role in regulating the severity of acute pancreatitis and pancreatitis-associated lung injury.
Monosomy of a specific chromosome determines
l
-sorbose utilization: A novel regulatory mechanism in
Candida albicans
Authors: Janbon Guilhem , Sherman Fred , Rustchenko Elena
Published: July 2002

We report the identification of the gene,
SOU1
, required for
l
-sorbose assimilation in
Candida albicans
. The level of the expression of
SOU1
is determined by the copy number of chromosome III
(also denoted chromosome 5), such that monosomic strains assimilate
l
-sorbose, whereas disomic strains do not, in spite of the
fact that
SOU1
is not on this chromosome. We suggest
that
C. albicans
contains a resource of potentially
beneficial genes that are activated by changes in chromosome number,
and that this elaborate mechanism regulates the utilization of food
supplies and possibly other important functions, thus representing a
novel general means for regulating gene expression in microbes.
Myocardial infarction mediated by endothelin receptor signaling in hypercholesterolemic mice
Authors: Caligiuri Giuseppina , Levy Bernard , Pernow John , Thorén Peter , Hansson Göran K.
Published: July 2002

Myocardial infarction is linked to atherosclerosis, yet the sequence leading from silent coronary atherosclerosis to acute myocardial infarction has remained unclear. Here we show that hypercholesterolemic apolipoprotein E
−/−
low density lipoprotein receptor
−/−
mice develop not only coronary atherosclerosis but also myocardial infarction. Exposure of mice to mental stress or hypoxia led to acute ischemia, which, in a large proportion of the mice, was followed by electrocardiographic changes, leakage of troponin T, and loss of dehydrogenase from the myocardium, all indicative of acute myocardial infarction. Apoptotic death of cardiomyocytes was followed by inflammation and fibrosis in the heart. All these pathological changes could be prevented by a blocker of the endothelin type A receptor. Thus, stress elicits myocardial infarction through endothelin receptor signaling in coronary atherosclerosis caused by hypercholesterolemia.
Identification of human prostaglandin E synthase: A microsomal, glutathione-dependent, inducible enzyme, constituting a potential novel drug target
Authors: Jakobsson Per-Johan , Thorén Staffan , Morgenstern Ralf , Samuelsson Bengt
Published: July 2002

Human prostaglandin (PG) E synthase (EC5.3.99.3) is a member of a recently recognized protein superfamily consisting of membrane associated proteins involved in eicosanoid and glutathione metabolism (the MAPEG family). Previous designations of the protein are PIG12 and MGST1-L1. PGE synthase was expressed inEscherichia coli, and both cytosolic and membrane fractions were prepared. Western blot analysis specifically detected a 15- to 16-kDa protein in the membrane fraction. Both fractions were incubated with prostaglandin H2in the presence or absence of reduced glutathione. The membrane but not the cytosolic fraction was found to possess high glutathione-dependent PGE synthase activity (0.25 μmol/min/mg). The human tissue distribution was analyzed by Northern blot analysis. High expression of PGE synthase mRNA was detected in A549 and HeLa cancer cell lines. Intermediate level of expression was demonstrated in placenta, prostate, testis, mammary gland, and bladder whereas low mRNA expression was observed in several other tissues. A549 cells have been used as a model system to study cyclooxygenase-2 induction by IL-1β. If A549 cells were grown in the presence of IL-1β, a significant induction of the PGE synthase was observed by Western blot analysis. Also, Western blot analysis specifically detected a 16-kDa protein in sheep seminal vesicles. In summary, we have identified a human membrane bound PGE synthase. The enzyme activity is glutathione-dependent, and the protein expression is induced by the proinflammatory cytokine IL-1β. PGE synthase is a potential novel target for drug development.
The natriuretic peptide clearance receptor locally modulates the physiological effects of the natriuretic peptide system
Authors: Matsukawa Naomichi , Grzesik Wojciech J. , Takahashi Nobuyuki , Pandey Kailash N. , Pang Stephen , Yamauchi Mitsuo , Smithies Oliver
Published: July 2002

Natriuretic peptides (NPs), mainly produced in heart [atrial (ANP) and B-type (BNP)], brain (CNP), and kidney (urodilatin), decrease blood pressure and increase salt excretion. These functions are mediated by natriuretic peptide receptors A and B (NPRA and NPRB) having cytoplasmic guanylyl cyclase domains that are stimulated when the receptors bind ligand. A more abundantly expressed receptor (NPRC or C-type) has a short cytoplasmic domain without guanylyl cyclase activity. NPRC is thought to act as a clearance receptor, although it may have additional functions. To test how NPRC affects the cardiovascular and renal systems, we inactivated its gene (
Npr3
) in mice by homologous recombination. The half life of [
125
I]ANP in the circulation of homozygotes lacking NPRC is two-thirds longer than in the wild type, although plasma levels of ANP and BNP in heterozygotes and homozygotes are close to the wild type. Heterozygotes and homozygotes have a progressively reduced ability to concentrate urine, exhibit mild diuresis, and tend to be blood volume depleted. Blood pressure in the homozygotes is 8 mmHg (1 mmHg = 133 Pa) below normal. These results are consistent with the sole cardiovascular/renal function of NPRC being to clear natriuretic peptides, thereby modulating local effects of the natriuretic peptide system. Unexpectedly,
Npr3
−/− homozygotes have skeletal deformities associated with a considerable increase in bone turnover. The phenotype is consistent with the bone function of NPRC being to clear locally synthesized CNP and modulate its effects. We conclude that NPRC modulates the availability of the natriuretic peptides at their target organs, thereby allowing the activity of the natriuretic peptide system to be tailored to specific local needs.
Bax-induced cell death in tobacco is similar to the hypersensitive response
Authors: Lacomme Christophe , Santa Cruz Simon
Published: July 2002

Bax, a death-promoting member of the Bcl-2 family of proteins, triggered cell death when expressed in plants from a tobacco mosaic virus vector. Analysis of Bax deletion mutants demonstrated a requirement for the BH1 and BH3 domains in promoting rapid cell death, whereas deletion of the carboxyl-terminal transmembrane domain completely abolished the lethality of Bax in plants. The phenotype of cell death induced by Bax closely resembled the hypersensitive response induced by wild-type tobacco mosaic virus in tobacco plants carrying the
N
gene. The cell death-promoting function of Bax in plants correlated with accumulation of the defense-related protein PR1, suggesting Bax activated an endogenous cell-death program in plants. In support of this view, both
N
gene- and Bax-mediated cell death was blocked by okadaic acid, an inhibitor of protein phosphatase activity. The ability of Bax to induce cell death and a defense reaction in plants suggests that some features of animal and plant cell death processes may be shared.
An unusual structural motif of antimicrobial peptides containing end-to-end macrocycle and cystine-knot disulfides
Authors: Tam James P. , Lu Yi-An , Yang Jin-Long , Chiu Kou-Wei
Published: July 2002

Four macrocyclic cystine-knot peptides of 29–31 residues, kalata, circulin A and B (CirA and CirB), and cyclopsychotride, have been isolated from coffee plants but have undetermined physiological functions. These macrocycles and 10 of their analogs prepared by chemical synthesis were tested against nine strains of microbes. Kalata and CirA were specific for the Gram-positiveStaphylococcus aureuswith a minimum inhibition concentration of ≈0.2 μM. They were relatively ineffective against Gram-negative bacteria such asEscherichia coliandPseudomonas aeruginosa. However, CirB and cyclopsychotride were active against both Gram-positive and Gram-negative bacteria. In particular, CirB showed potent activity againstE. coliwith a minimum inhibitory concentration of 0.41 μM. All four cyclic peptides were moderately active against two strains of fungi,Candida kefyrandCandida tropicalis, but were inactive againstCandida albicans. These macrocycles are cytotoxic and lysed human red blood cell with a lethal dose 50% of 400 μM. Modifying the Arg residue in kalata with a keto aldehyde significantly reduced its activity againstS. aureuswhereas blocking the arg in CirA produced no significant effect. The two-disulfide variants and their scrambled disulfide isomers exhibited antimicrobial profiles and potency similar to their native peptides. However, in high-salt assays (100 mM NaCl), few of these macrocyclic peptides, natives or analogs, retained antimicrobial activity. These results show that the macrocyclic peptides possess specific and potent antimicrobial activity that is salt-dependent and that their initial interactions with the microbial surfaces may be electrostatic, an effect commonly found in defensin antimicrobial peptides. Furthermore, their end-to-end cyclic structure with a cystine-knot motif represents a molecular structure of antimicrobials and may provide a useful template for the design of novel peptide antibiotics.
A vital role for glycosphingolipid synthesis during development and differentiation
Authors: Yamashita Tadashi , Wada Ryuichi , Sasaki Teiji , Deng Chuxia , Bierfreund Uwe , Sandhoff Konrad , Proia Richard L.
Published: July 2002

Glycosphingolipids (GSLs) are believed to be integral for the dynamics of many cell membrane events, including cellular interactions, signaling, and trafficking. We have investigated their roles in development and differentiation by eliminating the major synthesis pathway of GSLs through targeted disruption of the
Ugcg
gene encoding glucosylceramide synthase. In the absence of GSL synthesis, embryogenesis proceeded well into gastrulation with differentiation into primitive germ layers and patterning of the embryo but was abruptly halted by a major apoptotic process.
In vivo
, embryonic stem cells deficient in GSL synthesis were again able to differentiate into endodermal, mesodermal, and ectodermal derivatives but were strikingly deficient in their ability to form well differentiated tissues.
In vitro
, however, hematopoietic and neuronal differentiation could be induced. The results demonstrate that the synthesis of GSL structures is essential for embryonic development and for the differentiation of some tissues and support the concept that GSLs are involved in crucial cell interactions mediating these processes.
Distinctive gene expression patterns in human mammary epithelial cells and breast cancers
Authors: Perou Charles M. , Jeffrey Stefanie S. , van de Rijn Matt , Rees Christian A. , Eisen Michael B. , Ross Douglas T. , Pergamenschikov Alexander , Williams Cheryl F. , Zhu Shirley X. , Lee Jeffrey C. F. , Lashkari Deval , Shalon Dari , Brown Patrick O. , Botstein David
Published: July 2002

cDNA microarrays and a clustering algorithm were used to identify patterns of gene expression in human mammary epithelial cells growing in culture and in primary human breast tumors. Clusters of coexpressed genes identified through manipulations of mammary epithelial cellsin vitroalso showed consistent patterns of variation in expression among breast tumor samples. By using immunohistochemistry with antibodies against proteins encoded by a particular gene in a cluster, the identity of the cell type within the tumor specimen that contributed the observed gene expression pattern could be determined. Clusters of genes with coherent expression patterns in cultured cells and in the breast tumors samples could be related to specific features of biological variation among the samples. Two such clusters were found to have patterns that correlated with variation in cell proliferation rates and with activation of the IFN-regulated signal transduction pathway, respectively. Clusters of genes expressed by stromal cells and lymphocytes in the breast tumors also were identified in this analysis. These results support the feasibility and usefulness of this systematic approach to studying variation in gene expression patterns in human cancers as a means to dissect and classify solid tumors.
Targeted disruption ofTraf5gene causes defects in CD40- and CD27-mediated lymphocyte activation
Authors: Nakano Hiroyasu , Sakon Sachiko , Koseki Haruhiko , Takemori Toshitada , Tada Kurisu , Matsumoto Mitsuru , Munechika Eiko , Sakai Tsuyoshi , Shirasawa Takuji , Akiba Hisaya , Kobata Tetsuji , Santee Sybil M. , Ware Carl F. , Rennert Paul D. , Taniguchi Masaru , Yagita Hideo , Okumura Ko
Published: July 2002

TRAF5 [tumor necrosis factor (TNF) receptor-associated factor 5] is implicated in NF-κB and c-Jun NH2-terminal kinase/stress-activated protein kinase activation by members of the TNF receptor superfamily, including CD27, CD30, CD40, and lymphotoxin-β receptor. To investigate the functional role of TRAF5in vivo, we generated TRAF5-deficient mice by gene targeting. Activation of either NF-κB or c-Jun NH2-terminal kinase/stress-activated protein kinase by tumor necrosis factor, CD27, and CD40 was not abrogated intraf5−/−mice. However,traf5−/−B cells showed defects in proliferation and up-regulation of various surface molecules, including CD23, CD54, CD80, CD86, and Fas in response to CD40 stimulation. Moreover,in vitroIg production oftraf5−/−B cells stimulated with anti-CD40 plus IL-4 was reduced substantially. CD27-mediated costimulatory signal also was impaired intraf5−/−T cells. Collectively, these results demonstrate that TRAF5 is involved in CD40- and CD27-mediated signaling.
CoREST: A functional corepressor required for regulation of neural-specific gene expression
Authors: Andrés María E. , Burger Corinna , Peral-Rubio María J. , Battaglioli Elena , Anderson Mary E. , Grimes Julia , Dallman Julia , Ballas Nurit , Mandel Gail
Published: July 2002

Several genes encoding proteins critical to the neuronal phenotype, such as the brain type II sodium channel gene, are expressed to high levels only in neurons. This cell specificity is due, in part, to long-term repression in nonneural cells mediated by the repressor protein REST/NRSF (RE1 silencing transcription factor/neural-restrictive silencing factor). We show here that CoREST, a newly identified human protein, functions as a corepressor for REST. A single zinc finger motif in REST is required for CoREST interaction. Mutations of the motif that disrupt binding also abrogate repression. When fused to a Gal4 DNA-binding domain, CoREST functions as a repressor. CoREST is present in cell lines that express REST, and the proteins are found in the same immunocomplex. CoREST contains two SANT (SW13/ADA2/NCoR/TFIIIB B) domains, a structural feature of the nuclear receptor and silencing mediator for retinoid and thyroid human receptors (SMRT)-extended corepressors that mediate inducible repression by steroid hormone receptors. Together, REST and CoREST mediate repression of the type II sodium channel promoter in nonneural cells, and the REST/CoREST complex may mediate long-term repression essential to maintenance of cell identity.
Bacterial inactivation by using near- and supercritical carbon dioxide
Authors: Dillow Angela K. , Dehghani Fariba , Hrkach Jeffrey S. , Foster Neil R. , Langer Robert
Published: July 2002

The three most common methods of sterilization in use today are ethylene oxide exposure, γ-irradiation, and steam sterilization. Each of these methods has serious limitations for the sterilization of some materials used in medicine, especially thermally and hydrolytically sensitive polymers by themselves and in combination with proteins. In this work, we demonstrate a potential new method of sterilization by using supercritical fluid carbon dioxide. Using this method we achieve complete inactivation of a wide variety of bacterial organisms at moderate temperatures and in the absence of organic solvents or irradiation. Sterilization is a function of both the proximity to the fluid’s critical point and the chemical nature of the fluid itself. When biodegradable polymers poly(lactic-
co-
glycolic) acid and polylactic acid were included in the sterilization process, there was no effect on the inactivation efficiency, yet no physical or chemical damage to these thermally and hydrolytically labile materials was observed.
De novo
amyloid proteins from designed combinatorial libraries
Authors: West Michael W. , Wang Weixun , Patterson Jennifer , Mancias Joseph D. , Beasley James R. , Hecht Michael H.
Published: July 2002

Amyloid deposits are associated with several neurodegenerative diseases, including Alzheimer’s disease and the prion diseases. The amyloid fibrils isolated from these different diseases share similar structural features. However, the protein sequences that assemble into these fibrils differ substantially from one disease to another. To probe the relationship between amino acid sequence and the propensity to form amyloid, we studied a combinatorial library of sequences designed
de novo
. All sequences in the library were designed to share an identical pattern of alternating polar and nonpolar residues, but the precise identities of these side chains were not constrained and were varied combinatorially. The resulting proteins self-assemble into large oligomers visible by electron microscopy as amyloid-like fibrils. Like natural amyloid, the
de novo
fibrils are composed of β-sheet secondary structure and bind the diagnostic dye, Congo red. Thus, binary patterning of polar and nonpolar residues arranged in alternating periodicity can direct protein sequences to form fibrils resembling amyloid. The model amyloid fibrils assemble and disassemble reversibly, providing a tractable system for both basic studies into the mechanisms of fibril assembly and the development of molecular therapies that interfere with this assembly.
Late maturation of visual spatial integration in humans
Authors: Kovács Ilona , Kozma Petra , Fehér Ákos , Benedek György
Published: July 2002

Visual development is thought to be completed at an early age. We suggest that the maturation of the visual brain is not homogeneous: functions with greater need for early availability, such as visuomotor control, mature earlier, and the development of other visual functions may extend well into childhood. We found significant improvement in children between 5 and 14 years in visual spatial integration by using a contour-detection task. The data show that long-range spatial interactions—subserving the integration of orientational information across the visual field—span a shorter spatial range in children than in adults. Performance in the task improves in a cue-specific manner with practice, which indicates the participation of fairly low-level perceptual mechanisms. We interpret our findings in terms of a protracted development of ventral visual-stream function in humans.
c-Myc enhances protein synthesis and cell size during B lymphocyte development
Authors: Iritani Brian M. , Eisenman Robert N.
Published: July 2002

Members of themycfamily of nuclear protooncogenes play roles in cell proliferation, differentiation, and apoptosis. Moreover, inappropriate expression of c-mycgenes contributes to the development of many types of cancers, including B cell lymphomas in humans. Although Myc proteins have been shown to function as transcription factors, their immediate effects on the cell have not been well defined. Here we have utilized a murine model of lymphomagenesis (Eμ-mycmice) to show that constitutive expression of a c-myctransgene under control of the Ig heavy-chain enhancer (Eμ) results in an increase in cell size of normal pretransformed B lymphocytes at all stages of B cell development. Furthermore, we show that c-Myc-induced growth occurs independently of cell cycle phase and correlates with an increase in protein synthesis. These results suggest that Myc may normally function by coordinating expression of growth-related genes in response to mitogenic signals. Deregulated c-mycexpression may predispose to cancer by enhancing cell growth to levels required for unrestrained cell division.
NMR spectroscopy in studies of light-induced structural changes in mammalian rhodopsin: Applicability of solution
19
F NMR
Authors: Klein-Seetharaman Judith , Getmanova Elena V. , Loewen Michèle C. , Reeves Philip J. , Khorana H. Gobind
Published: July 2002

We report high resolution solution
19
F NMR spectra of fluorine-labeled rhodopsin mutants in detergent micelles. Single cysteine substitution mutants in the cytoplasmic face of rhodopsin were labeled by attachment of the trifluoroethylthio (TET), CF
3
-CH
2
-S, group through a disulfide linkage. TET-labeled cysteine mutants at amino acid positions 67, 140, 245, 248, 311, and 316 in rhodopsin were thus prepared. Purified mutant rhodopsins (6–10 mg), in dodecylmaltoside, were analyzed at 20°C by solution
19
F NMR spectroscopy. The spectra recorded in the dark showed the following chemical shifts relative to trifluoroacetate: Cys-67, 9.8 ppm; Cys-140, 10.6 ppm; Cys-245, 9.9 ppm; Cys-248, 9.5 ppm; Cys-311, 9.9 ppm; and Cys-316, 10.0 ppm. Thus, all mutants showed chemical shifts downfield that of free TET (6.5 ppm). On illumination to form metarhodopsin II, upfield changes in chemical shift were observed for
19
F labels at positions 67 (−0.2 ppm) and 140 (−0.4 ppm) and downfield changes for positions 248 (+0.1 ppm) and 316 (+0.1 ppm) whereas little or no change was observed at positions 311 and 245. On decay of metarhodopsin II, the chemical shifts reverted largely to those originally observed in the dark. The results demonstrate the applicability of solution
19
F NMR spectroscopy to studies of the tertiary structures in the cytoplasmic face of intact rhodopsin in the dark and on light activation.
Variable efficacy of repeated annual influenza vaccination
Authors: Smith Derek J. , Forrest Stephanie , Ackley David H. , Perelson Alan S.
Published: July 2002

Conclusions have differed in studies that have compared vaccine efficacy in groups receiving influenza vaccine for the first time to efficacy in groups vaccinated more than once. For example, the Hoskins study [Hoskins, T. W., Davis, J. R., Smith, A. J., Miller, C. L. & Allchin, A. (1979)
Lancet
i, 33–35] concluded that repeat vaccination was not protective in the long term, whereas the Keitel study [Keitel, W. A., Cate, T. R., Couch, R. B., Huggins, L. L. & Hess, K. R. (1997)
Vaccine
15, 1114–1122] concluded that repeat vaccination provided continual protection. We propose an explanation, the
antigenic distance
hypothesis, and test it by analyzing seven influenza outbreaks that occurred during the Hoskins and Keitel studies. The hypothesis is that variation in repeat vaccine efficacy is due to differences in antigenic distances among vaccine strains and between the vaccine strains and the epidemic strain in each outbreak. To test the hypothesis, antigenic distances were calculated from historical hemagglutination inhibition assay tables, and a computer model of the immune response was used to predict the vaccine efficacy of individuals given different vaccinations. The model accurately predicted the observed vaccine efficacies in repeat vaccinees relative to the efficacy in first-time vaccinees (correlation 0.87). Thus, the antigenic distance hypothesis offers a parsimonious explanation of the differences between and within the Hoskins and Keitel studies. These results have implications for the selection of influenza vaccine strains, and also for vaccination strategies for other antigenically variable pathogens that might require repeated vaccination.