bluuebunny/crossref_metadata_2025 huggingface dataset:
Inhibiting transthyretin amyloid fibril formation via protein stabilization
Authors: Miroy Greta J. , Lai Zhihong , Lashuel Hilal A. , Peterson Scott A. , Strang Candace , Kelly Jeffery W.
Published: July 2002

Transthyretin (TTR) amyloid fibril formation is observed
systemically in familial amyloid polyneuropathy and senile systemic
amyloidosis and appears to be the causative agent in these diseases.
Herein, we demonstrate conclusively that thyroxine (10.8 μM) inhibits
TTR fibril formation efficiently
in vitro
and does so by
stabilizing the tetramer against dissociation and the subsequent
conformational changes required for amyloid fibril formation. In
addition, the nonnative ligand 2,4,6-triiodophenol, which binds to TTR
with slightly increased affinity also inhibits TTR fibril formation by
this mechanism. Sedimentation velocity experiments were employed to
show that TTR undergoes dissociation (linked to a conformational
change) to form the monomeric amyloidogenic intermediate, which
self-assembles into amyloid in the absence, but not in the presence of
thyroxine. These results demonstrate the feasibility of using small
molecules to stabilize the native fold of a potentially amyloidogenic
human protein, thus preventing the conformational changes, which appear
to be the common link in several human amyloid diseases. This strategy
and the compounds resulting from further development should prove
useful for critically evaluating the amyloid hypothesis—i.e., the
putative cause-and-effect relationship between TTR amyloid deposition
and the onset of familial amyloid polyneuropathy and senile systemic
amyloidosis.
Prevention of hepatitis C virus infection in chimpanzees by hyperimmune serum against the hypervariable region 1 of the envelope 2 protein
Authors: Farci Patrizia , Shimoda Atsushi , Wong Doris , Cabezon Teresa , De Gioannis Daniela , Strazzera Antonello , Shimizu Yohko , Shapiro Max , Alter Harvey J. , Purcell Robert H.
Published: July 2002

The identification of the neutralization domains of hepatitis C
virus (HCV) is essential for the development of an effective vaccine.
Here, we show that the hypervariable region 1 (HVR1) of the envelope 2
(E2) protein is a critical neutralization domain of HCV. Neutralization
of HCV
in vitro
was attempted with a rabbit hyperimmune
serum raised against a homologous synthetic peptide derived from the
HVR1 of the E2 protein, and the residual infectivity was evaluated by
inoculation of HCV-seronegative chimpanzees. The source of HCV was
plasma obtained from a patient (H) during the acute phase of
posttransfusion non-A, non-B hepatitis, which had been titered for
infectivity in chimpanzees. The anti-HVR1 antiserum induced protection
against homologous HCV infection in chimpanzees, but not against the
emergence of neutralization escape mutants that were found to be
already present in the complex viral quasispecies of the inoculum. The
finding that HVR1 can elicit protective immunity opens new perspectives
for the development of effective preventive strategies. However, the
identification of the most variable region of HCV as a critical
neutralization domain poses a major challenge for the development of a
broadly reactive vaccine against HCV.
Embryonic expression patterns of the neural cell adhesion molecule gene are regulated by homeodomain binding sites.
Authors: Wang Y , Jones F S , Krushel L A , Edelman G M
Published: July 2002

During development of the vertebrate nervous system, the neural cell adhesion molecule (N-CAM) is expressed in a defined spatiotemporal pattern. We have proposed that the expression of N-CAM is controlled, in part, by proteins encoded by homeobox genes. This hypothesis has been supported by previous in vitro experiments showing that products of homeobox genes can both bind to and transactivate the N-CAM promoter via two homeodomain binding sites, HBS-I and HBS-II. We have now tested the hypothesis that the N-CAM gene is a target of homeodomain proteins in vivo by using transgenic mice containing native and mutated N-CAM promoter constructs linked to a beta-galactosidase reporter gene. Segments of the 5' flanking region of the mouse N-CAM gene were sufficient to direct expression of the reporter gene in the central nervous system in a pattern consistent with that of the endogenous N-CAM gene. For example, at embryonic day (E) 11, beta-galactosidase staining was found in postmitotic neurons in dorsolateral and ventrolateral regions of the spinal cord; at E14.5, staining was seen in these neurons throughout the spinal cord. In contrast, mice carrying an N-CAM promoter-reporter construct with mutations in both homeodomain binding sites (HBS-I and HBS-II) showed altered expression patterns in the spinal cord. At E11, beta-galactosidase expression was seen in the ventrolateral spinal cord, but was absent in the dorsolateral areas, and at E 14.5, beta-galactosidase expression was no longer detected in any cells of the cord. Homeodomain binding sites found in the N-CAM promoter thus appear to be important in determining specific expression patterns of N-CAM along the dorsoventral axis in the developing spinal cord. These experiments suggest that the N-CAM gene is an in vivo target of homeobox gene products in vertebrates.
Prevention of diabetic alterations in transgenic mice overexpressing Myc in the liver.
Authors: Riu E , Bosch F , Valera A
Published: July 2002

Recent studies have demonstrated that the overexpression of the c-myc gene in the liver of transgenic mice leads to an increase in both utilization and accumulation of glucose in the liver, suggesting that c-Myc transcription factor is involved in the control of liver carbohydrate metabolism in vivo. To determine whether the increase in c-Myc might control glucose homeostasis, an intraperitoneal glucose tolerance test was performed. Transgenic mice showed lower levels of blood glucose than control animals, indicating that the overexpression of c-Myc led to an increase of blood glucose disposal by the liver. Thus, the increase in c-Myc might counteract diabetic hyperglycemia. In contrast to control mice, transgenic mice treated with streptozotocin showed normalization of concentrations of blood glucose, ketone bodies, triacylglycerols and free fatty acids in the absence of insulin. These findings resulted from the normalization of liver metabolism in these animals. While low glucokinase activity was detected in the liver of diabetic control mice, high levels of both glucokinase mRNA and enzyme activity were noted in the liver of streptozotocin-treated transgenic mice, which led to an increase in intracellular levels of glucose 6-phosphate and glycogen. The liver of these mice also showed an increase in pyruvate kinase activity and lactate production. Furthermore, normalization of both the expression of genes involved in the control of gluconeogenesis and ketogenesis and the production of glucose and ketone bodies was observed in streptozotocin-treated transgenic mice. Thus, these results suggested that c-Myc counteracted diabetic alterations through its ability to induce hepatic glucose uptake and utilization and to block the activation of gluconeogenesis and ketogenesis.
4-Hydroxylation of estrogens as marker of human mammary tumors.
Authors: Liehr J G , Ricci M J
Published: July 2002

Estrogen is a known risk factor in human breast cancer. In rodent models, estradiol has been shown to induce tumors in those tissues in which this hormone is predominantly converted to the catechol metabolite 4-hydroxyestradiol by a specific 4-hydroxylase enzyme, whereas tumors fail to develop in organs in which 2-hydroxylation predominates. We have now found that microsomes prepared from human mammary adenocarcinoma and fibroadenoma predominantly catalyze the metabolic 4-hydroxylation of estradiol (ratios of 4-hydroxyestradiol/2-hydroxyestradiol formation in adenocarcinoma and fibroadenoma, 3.8 and 3.7, respectively). In contrast, microsomes from normal tissue obtained either from breast cancer patients or from reduction mammoplasty operations expressed comparable estradiol 2- and 4-hydroxylase activities (corresponding ratios, 1.3 and 0.7, respectively). An elevated ratio of 4-/2-hydroxyestradiol formation in neoplastic mammary tissue may therefore provide a useful marker of benign or malignant breast tumors and may indicate a mechanistic role of 4-hydroxyestradiol in tumor development.
Aberrant retention of tyrosinase in the endoplasmic reticulum mediates accelerated degradation of the enzyme and contributes to the dedifferentiated phenotype of amelanotic melanoma cells
Authors: Halaban Ruth , Cheng Elaine , Zhang Yuhua , Moellmann Gisela , Hanlon Douglas , Michalak Marek , Setaluri Vijayasaradhi , Hebert Daniel N.
Published: July 2002

The loss of tyrosinase, the key enzyme in melanin synthesis, has been implicated in the dedifferentiation of malignant melanocytes. The presence of tyrosinase transcripts and antigenic peptides in melanoma tumors prompted us to investigate whether the basis for the loss of the enzyme was proteolytic degradation. Toward this aim, we followed the kinetics of synthesis, degradation, processing, chaperone binding, inhibitor sensitivity, and subcellular localization of tyrosinase in normal and malignant melanocytes. We found that, in amelanotic melanoma cell lines, tyrosinase failed to reach the melanosome, the organelle for melanin synthesis, because it was retained in the endoplasmic reticulum (ER) and then degraded. Tyrosinase appeared mostly as a 70-kDa core-glycosylated, endoglycosidase H-sensitive, immature form bound to the ER chaperone calnexin and had a life-span of only 25% of normal. Maturation and transit from the ER to the Golgi compartment was facilitated by lowering the temperature of incubation to 31°C. Several proteasome inhibitors caused the accumulation of an ≈60-kDa tyrosinase doublet that was more prominent in malignant than in normal melanocytes and promoted, to various degrees, the maturation of tyrosinase in melanoma cells and the translocation of the enzyme to melanosomes. The appearance of ubiquitinated tyrosinase after treatment of normal melanocytes withN-acetyl-l-leucinyl-l-leucinal-l-norleucinal reinforced our notion that some tyrosinase is normally degraded by proteasomes. Proteolysis of tyrosinase by proteasomes is consistent with the production of antigenic tyrosinase peptides that are presented to the immune system by major histocompatibility complex class I molecules.
Identification of the yeast 20S proteasome catalytic centers and subunit interactions required for active-site formation
Authors: Arendt Cassandra S. , Hochstrasser Mark
Published: July 2002

The proteasome is responsible for degradation of substrates of the ubiquitin pathway. 20S proteasomes are cylindrical particles with subunits arranged in a stack of four heptameric rings. The outer rings are composed of α subunits, and the inner rings are composed of β subunits. A well-characterized archaeal proteasome has a single type of each subunit, and the N-terminal threonine of the β subunit is the active-site nucleophile. Yeast proteasomes have seven different β subunits and exhibit several distinct peptidase activities, which were proposed to derive from disparate active sites. We show that mutating the N-terminal threonine in the yeast Pup1 β subunit eliminates cleavage after basic residues in peptide substrates, and mutating the corresponding threonine of Pre3 prevents cleavage after acidic residues. Surprisingly, neither mutation has a strong effect on cell growth, and they have at most minor effects on ubiquitin-dependent proteolysis. We show that Pup1 interacts with Pup3 in each β subunit ring. Our data reveal that different proteasome active sites contribute very differently to protein breakdown
in vivo
, that contacts between particular subunits in each β subunit ring are critical for active-site formation, and that active sites in archaea and different eukaryotes are highly similar.
Altered expression of the WT1 Wilms tumor suppressor gene in human breast cancer
Authors: Silberstein Gary B. , Van Horn Katharine , Strickland Phyllis , Roberts Charles T. , Daniel Charles W.
Published: July 2002

The product of the WT1 Wilms tumor suppressor gene controls the expression of genes encoding components of the insulin-like growth factor and transforming growth factor β signaling systems. The role of these growth factors in breast tumor growth led us to investigate possible WT1 gene expression in normal and cancerous breast tissue. WT1 was detected by immunohistochemistry in the normal mammary duct and lobule, and the patterns of expression were consistent with developmental regulation. In a survey of 21 infiltrating tumors, 40% lacked immunodetectable WT1 altogether and an additional 28% were primarily WT1-negative. Cytoplasmic, but not nuclear, localization of WT1 was noted in some tumor cells and WT1 was detected, sometimes at high levels, in more-advanced estrogen-receptor-negative tumors. In this highly malignant subset, the tumor suppressor protein p53, which can physically interact with WT1, was also sometimes detected. WT1 mRNA was detected in normal and tumor tissue by reverse transcription-coupled PCR. Alternative splicing of the WT1 mRNA may regulate gene targeting of the WT1 protein through changes either in its regulatory or zinc-finger domains. The relative proportions of WT1 mRNA splice variants were altered in a random sample of breast tumors, providing evidence that different tumors may share a common WT1-related defect resulting in altered regulation of target genes.
Engineering a tRNA and aminoacyl-tRNA synthetase for the site-specific incorporation of unnatural amino acids into proteins
in vivo
Authors: Liu David R. , Magliery Thomas J. , Pastrnak Miro , Schultz Peter G.
Published: July 2002

In an effort to expand the scope of protein mutagenesis, we have completed the first steps toward a general method to allow the site-specific incorporation of unnatural amino acids into proteins
in vivo
. Our approach involves the generation of an “orthogonal” suppressor tRNA that is uniquely acylated in
Escherichia coli
by an engineered aminoacyl-tRNA synthetase with the desired unnatural amino acid. To this end, eight mutations were introduced into tRNA
2
Gln
based on an analysis of the x-ray crystal structure of the glutaminyl-tRNA aminoacyl synthetase (GlnRS)–tRNA
2
Gln
complex and on previous biochemical data. The resulting tRNA satisfies the minimal requirements for the delivery of an unnatural amino acid: it is not acylated by any endogenous
E. coli
aminoacyl-tRNA synthetase including GlnRS, and it functions efficiently in protein translation. Repeated rounds of DNA shuffling and oligonucleotide-directed mutagenesis followed by genetic selection resulted in mutant GlnRS enzymes that efficiently acylate the engineered tRNA with glutamine
in vitro
. The mutant GlnRS and engineered tRNA also constitute a functional synthetase–tRNA pair
in vivo
. The nature of the GlnRS mutations, which occur both at the protein–tRNA interface and at sites further away, is discussed.
Compositional differences within and between eukaryotic genomes
Authors: Karlin Samuel , Mrázek Jan
Published: July 2002

Eukaryotic genome similarity relationships are inferred using sequence information derived from large aggregates of genomic sequences. Comparisons within and between species sample sequences are based on the profile of dinucleotide relative abundance values (The profile is ρ*

XY

=
f
*

XY

/
f
*

X

f
*

Y

for all
XY
, where
f
*

X

denotes the frequency of the nucleotide
X
and
f
*

XY

denotes the frequency of the dinucleotide
XY
, both computed from the sequence concatenated with its inverted complement). Previous studies with respect to prokaryotes and this study document that profiles of different DNA sequence samples (sample size ≥50 kb) from the same organism are generally much more similar to each other than they are to profiles from other organisms, and that closely related organisms generally have more similar profiles than do distantly related organisms. On this basis we refer to the collection {ρ*

XY

} as the
genome signature
. This paper identifies ρ*

XY

extremes and compares genome signature differences for a diverse range of eukaryotic species. Interpretations on the mechanisms maintaining these profile differences center on genome-wide replication, repair, DNA structures, and context-dependent mutational biases. It is also observed that mitochondrial genome signature differences between species parallel the corresponding nuclear genome signature differences despite large differences between corresponding mitochondrial and nuclear signatures. The genome signature differences also have implications for contrasts between rodents and other mammals, and between monocot and dicot plants, as well as providing evidence for similarities among fungi and the diversity of protists.
Parametrizations of elliptic curves by Shimura curves and by classical modular curves
Authors: Ribet Kenneth A. , Takahashi Shuzo
Published: July 2002

Fix an isogeny class 𝒜 of semistable elliptic curves over Q. The
elements of 𝒜 have a common conductor
N
, which is a
square-free positive integer. Let
D
be a divisor of
N
which is the product of an even number of primes—i.e.,
the discriminant of an indefinite quaternion algebra over Q. To
D
we associate a certain Shimura curve
X
0

D

(
N
/
D
),
whose Jacobian is isogenous to an abelian subvariety of
J
0
(
N
). There is a unique
A
∈ 𝒜 for which one has a nonconstant map
π

D

:
X
0

D

(
N
/
D
)
→
A
whose pullback
A
→
Pic
0
(
X
0

D

(
N
/
D
))
is injective. The degree of π

D

is an integer
δ

D

which depends only on
D
(and
the fixed isogeny class 𝒜). We investigate the behavior of
δ

D

as
D
varies.
The cysteine-rich frizzled domain of Frzb-1 is required and sufficient for modulation of Wnt signaling
Authors: Lin Keming , Wang Shouwen , Julius Martin A. , Kitajewski Jan , Moos Malcolm , Luyten Frank P.
Published: July 2002

Convincing evidence has accumulated to identify the Frizzled proteins as receptors for the Wnt growth factors. In parallel, a number of secreted frizzled-like proteins with a conserved N-terminal frizzled motif have been identified. One of these proteins, Frzb-1, binds Wnt-1 and Xwnt-8 proteins and antagonizes Xwnt-8 signaling in
Xenopus
embryos. Here we report that Frzb-1 blocks Wnt-1 induced cytosolic accumulation of β-catenin, a key component of the Wnt signaling pathway, in human embryonic kidney cells. Structure/function analysis reveals that complete removal of the frizzled domain of Frzb-1 abolishes the Wnt-1/Frzb-1 protein interaction and the inhibition of Wnt-1 mediated axis duplication in
Xenopus
embryos. In contrast, removal of the C-terminal portion of the molecule preserves both Frzb-Wnt binding and functional inhibition of Wnt signaling. Partial deletions of the Frzb-1 cysteine-rich domain maintain Wnt-1 interaction, but functional inhibition is lost. Taken together, these findings support the conclusion that the frizzled domain is necessary and sufficient for both activities. Interestingly, Frzb-1 does not block Wnt-5A signaling in a
Xenopus
functional assay, even though Wnt-5A coimmunoprecipitates with Frzb-1, suggesting that coimmunoprecipitation does not necessarily imply inhibition of Wnt function.
Activation of protein kinase C by tyrosine phosphorylation in response to H
2
O
2
Authors: Konishi Hiroaki , Tanaka Motonari , Takemura Yukitoshi , Matsuzaki Hidenori , Ono Yoshitaka , Kikkawa Ushio , Nishizuka Yasutomi
Published: July 2002

Protein kinase C (PKC) isoforms, α, βI, and γ of cPKC subgroup, δ and ɛ of nPKC subgroup, and ζ of aPKC subgroup, were tyrosine phosphorylated in COS-7 cells in response to H
2
O
2
. These isoforms isolated from the H
2
O
2
-treated cells showed enhanced enzyme activity to various extents. The enzymes, PKC α and δ, recovered from the cells were independent of lipid cofactors for their catalytic activity. Analysis of mutated molecules of PKC δ showed that tyrosine residues, which are conserved in the catalytic domain of the PKC family, are critical for PKC activation induced by H
2
O
2
. These results suggest that PKC isoforms can be activated through tyrosine phosphorylation in a manner unrelated to receptor-coupled hydrolysis of inositol phospholipids.
Phosphorylation of two regulatory tyrosine residues in the activation of Bruton’s tyrosine kinase via alternative receptors
Authors: Wahl Matthew I. , Fluckiger Anne-Catherine , Kato Roberta M. , Park Hyunsun , Witte Owen N. , Rawlings David J.
Published: July 2002

Mutation of Bruton’s tyrosine kinase (Btk) impairs B cell maturation and function and results in a clinical phenotype of X-linked agammaglobulinemia. Activation of Btk correlates with an increase in the phosphorylation of two regulatory Btk tyrosine residues. Y551 (site 1) within the Src homology type 1 (SH1) domain is transphosphorylated by the Src family tyrosine kinases. Y223 (site 2) is an autophosphorylation site within the Btk SH3 domain. Polyclonal, phosphopeptide-specific antibodies were developed to evaluate the phosphorylation of Btk sites 1 and 2. Crosslinking of the B cell antigen receptor (BCR) or the mast cell Fcɛ receptor, or interleukin 5 receptor stimulation each induced rapid phosphorylation at Btk sites 1 and 2 in a tightly coupled manner. Btk molecules were singly and doubly tyrosine-phosphorylated. Phosphorylated Btk comprised only a small fraction (≤5%) of the total pool of Btk molecules in the BCR-activated B cells. Increased dosage of Lyn in B cells augmented BCR-induced phosphorylation at both sites. Kinetic analysis supports a sequential activation mechanism in which individual Btk molecules undergo serial transphosphorylation (site 1) then autophosphorylation (site 2), followed by successive dephosphorylation of site 1 then site 2. The phosphorylation of conserved tyrosine residues within structurally related Tec family kinases is likely to regulate their activation.
A mouse model for the renal salt-wasting syndrome pseudohypoaldosteronism
Authors: Hummler Edith , Barker Pierre , Talbot Colleen , Wang Qing , Verdumo Chantal , Grubb Barbara , Gatzy John , Burnier Michel , Horisberger Jean-Daniel , Beermann Friedrich , Boucher Richard , Rossier Bernard C.
Published: July 2002

Aldosterone-dependent epithelial sodium transport in the distal nephron is mediated by the absorption of sodium through the highly selective, amiloride-sensitive epithelial sodium channel (ENaC) made of three homologous subunits (α, β, and γ). In human, autosomal recessive mutations of α, β, or γENaC subunits cause pseudohypoaldosteronism type 1 (PHA-1), a renal salt-wasting syndrome characterized by severe hypovolemia, high plasma aldosterone, hyponatremia, life-threatening hyperkaliemia, and metabolic acidosis. In the mouse, inactivation of αENaC results in failure to clear fetal lung liquid at birth and in early neonatal death, preventing the observation of a PHA-1 renal phenotype. Transgenic expression of αENaC driven by a cytomegalovirus promoter in αENaC(−/−) knockout mice [αENaC(−/−)Tg] rescued the perinatal lethal pulmonary phenotype and partially restored Na
+
transport in renal, colonic, and pulmonary epithelia. At days 5–9, however, αENaC(−/−)Tg mice showed clinical features of severe PHA-1 with metabolic acidosis, urinary salt-wasting, growth retardation, and 50% mortality. Adult αENaC(−/−)Tg survivors exhibited a compensated PHA-1 with normal acid/base and electrolyte values but 6-fold elevation of plasma aldosterone compared with wild-type littermate controls. We conclude that partial restoration of ENaC-mediated Na
+
absorption in this transgenic mouse results in a mouse model for PHA-1.
Origin and evolution of the slime molds (Mycetozoa)
Authors: Baldauf Sandra L. , Doolittle W. Ford
Published: July 2002

The Mycetozoa include the cellular (dictyostelid), acellular (myxogastrid), and protostelid slime molds. However, available molecular data are in disagreement on both the monophyly and phylogenetic position of the group. Ribosomal RNA trees show the myxogastrid and dictyostelid slime molds as unrelated early branching lineages, but actin and β-tubulin trees place them together as a single coherent (monophyletic) group, closely related to the animal–fungal clade. We have sequenced the elongation factor-1α genes from one member of each division of the Mycetozoa, including
Dictyostelium discoideum
, for which cDNA sequences were previously available. Phylogenetic analyses of these sequences strongly support a monophyletic Mycetozoa, with the myxogastrid and dictyostelid slime molds most closely related to each other. All phylogenetic methods used also place this coherent Mycetozoan assemblage as emerging among the multicellular eukaryotes, tentatively supported as more closely related to animals + fungi than are green plants. With our data there are now three proteins that consistently support a monophyletic Mycetozoa and at least four that place these taxa within the “crown” of the eukaryote tree. We suggest that ribosomal RNA data should be more closely examined with regard to these questions, and we emphasize the importance of developing multiple sequence data sets.
Identification of a second aryl phosphate-binding site in protein-tyrosine phosphatase 1B: A paradigm for inhibitor design
Authors: Puius Yoram A. , Zhao Yu , Sullivan Michael , Lawrence David S. , Almo Steven C. , Zhang Zhong-Yin
Published: July 2002

The structure of the catalytically inactive mutant (C215S) of the human protein-tyrosine phosphatase 1B (PTP1B) has been solved to high resolution in two complexes. In the first, crystals were grown in the presence of bis-(
para
-phosphophenyl) methane (BPPM), a synthetic high-affinity low-molecular weight nonpeptidic substrate (
K
m
= 16 μM), and the structure was refined to an
R
-factor of 18.2% at 1.9 Å resolution. In the second, crystals were grown in a saturating concentration of phosphotyrosine (pTyr), and the structure was refined to an
R
-factor of 18.1% at 1.85 Å. Difference Fourier maps showed that BPPM binds PTP1B in two mutually exclusive modes, one in which it occupies the canonical pTyr-binding site (the active site), and another in which a phosphophenyl moiety interacts with a set of residues not previously observed to bind aryl phosphates. The identification of a second pTyr molecule at the same site in the PTP1B/C215S–pTyr complex confirms that these residues constitute a low-affinity noncatalytic aryl phosphate-binding site. Identification of a second aryl phosphate binding site adjacent to the active site provides a paradigm for the design of tight-binding, highly specific PTP1B inhibitors that can span both the active site and the adjacent noncatalytic site. This design can be achieved by tethering together two small ligands that are individually targeted to the active site and the proximal noncatalytic site.
Glucocorticoid-mediated repression of nuclear factor-κBdependent transcription involves direct interference with transactivation
Authors: De Bosscher Karolien , Schmitz M. Lienhard , Vanden Berghe Wim , Plaisance Stéphane , Fiers Walter , Haegeman Guy
Published: July 2002

Glucocorticoids exert multiple anti-inflammatory activities, one of which is the inhibition of transcription dependent on the nuclear factor (NF)-κB. It has been suggested that the effect of dexamethasone (DEX), a glucocorticoid analog, is attributed to an increased production of the inhibitory IκB molecule, which in turn would bind and remove activated, DNA-bound NF-κB complexes in the cell nucleus. Upon investigating DEX-mediated repression of interleukin-6 expression induced by tumor necrosis factor, DEX treatment was found to act directly on NF-κB-dependent transcription, without changing the expression level of IκB. Neither the mRNA of IκB nor the protein was significantly elevated by a combined treatment with tumor necrosis factor and DEX of murine endothelial or fibroblast cells. The DNA-binding activity of induced NF-κB also remained unchanged after stimulation of cells with DEX. Evidence for a direct nuclear mechanism of action was obtained by analysis of cell lines stably expressing a fusion protein between the DNA-binding domain of the yeast Gal4 protein and the transactivating p65 subunit of NF-κB. Expression of a Gal4-dependent luciferase reporter gene activated by this nuclear fusion protein was also strongly repressed after addition of DEX. Because the DNA-binding activity of the Gal4 fusion protein was not affected by DEX, it can be concluded that the reduction of gene activation was caused by interference of the activated glucocorticoid receptor with the transactivation potential of the NF-κB p65 subunit.
Tissue engineering of cartilage in space
Authors: Freed Lisa E. , Langer Robert , Martin Ivan , Pellis Neal R. , Vunjak-Novakovic Gordana
Published: July 2002

Tissue engineering of cartilage, i.e., the
in vitro
cultivation of cartilage cells on synthetic polymer scaffolds, was studied on the Mir Space Station and on Earth. Specifically, three-dimensional cell-polymer constructs consisting of bovine articular chondrocytes and polyglycolic acid scaffolds were grown in rotating bioreactors, first for 3 months on Earth and then for an additional 4 months on either Mir (10
−4
–10
−6
g
) or Earth (1
g
). This mission provided a unique opportunity to study the feasibility of long-term cell culture flight experiments and to assess the effects of spaceflight on the growth and function of a model musculoskeletal tissue. Both environments yielded cartilaginous constructs, each weighing between 0.3 and 0.4
g
and consisting of viable, differentiated cells that synthesized proteoglycan and type II collagen. Compared with the Earth group, Mir-grown constructs were more spherical, smaller, and mechanically inferior. The same bioreactor system can be used for a variety of controlled microgravity studies of cartilage and other tissues. These results may have implications for human spaceflight, e.g., a Mars mission, and clinical medicine, e.g., improved understanding of the effects of pseudo-weightlessness in prolonged immobilization, hydrotherapy, and intrauterine development.
Estrogen receptor-dependent sexual differentiation of dopaminergic neurons in the preoptic region of the mouse
Authors: Simerly R. B. , Zee M. C. , Pendleton J. W. , Lubahn D. B. , Korach K. S.
Published: July 2002

Although it has been known for some time that estrogen exerts a profound influence on brain development a definitive demonstration of the role of the classical estrogen receptor (ERα) in sexual differentiation has remained elusive. In the present study we used a sexually dimorphic population of dopaminergic neurons in the anteroventral periventricular nucleus of the hypothalamus (AVPV) to test the dependence of sexual differentiation on a functional ERα by comparing the number of tyrosine hydroxylase (TH)-immunoreactive neurons in the AVPV of wild-type (WT) mice with that of mice in which the ERα had been disrupted by homologous recombination (ERKOα). Only a few ERα-immunoreactive neurons were detected in the AVPV of ERKOα mice, and the number of TH-immunoreactive neurons was three times that of WT mice, suggesting that disruption of the ERα gene feminized the number of TH-immunoreactive neurons. In contrast, the AVPV contains the same number of TH-immunoreactive neurons in testicular feminized male mice as in WT males, indicating that sexual differentiation of this population of neurons is not dependent on an intact androgen receptor. The number of TH-immunoreactive neurons in the AVPV of female ERKOα mice remained higher than that of WT males, but TH staining appeared to be lower than that of WT females. Thus, the sexual differentiation of dopamine neurons in the AVPV appears to be receptor specific and dependent on the perinatal steroid environment.
Dynamic weighting in Monte Carlo and optimization
Authors: Wong Wing Hung , Liang Faming
Published: July 2002

Dynamic importance weighting is proposed as a Monte Carlo method that has the capability to sample relevant parts of the configuration space even in the presence of many steep energy minima. The method relies on an additional dynamic variable (the importance weight) to help the system overcome steep barriers. A non-Metropolis theory is developed for the construction of such weighted samplers. Algorithms based on this method are designed for simulation and global optimization tasks arising from multimodal sampling, neural network training, and the traveling salesman problem. Numerical tests on these problems confirm the effectiveness of the method.
Brain wave recognition of words
Authors: Suppes Patrick , Lu Zhong-Lin , Han Bing
Published: July 2002

Electrical and magnetic brain waves of seven subjects under three experimental conditions were recorded for the purpose of recognizing which one of seven words was processed. The analysis consisted of averaging over trials to create prototypes and test samples, to both of which Fourier transforms were applied, followed by filtering and an inverse transformation to the time domain. The filters used were optimal predictive filters, selected for each subject and condition. Recognition rates, based on a least-squares criterion, varied widely, but all but one of 24 were significantly different from chance. The two best were above 90%. These results show that brain waves carry substantial information about the word being processed under experimental conditions of conscious awareness.
Dissecting the thrombopoietin receptor: Functional elements of the Mpl cytoplasmic domain
Authors: Drachman Jonathan G. , Kaushansky Kenneth
Published: July 2002

Thrombopoietin (TPO) acts through its receptor, Mpl, to stimulate the proliferation and maturation of megakaryocytes and their progenitors. The Mpl cytoplasmic domain controls this process through assembly of an active signaling complex using various receptor docking sites. In this report, eight carboxyl truncations of the 121-aa murine Mpl cytoplasmic domain were tested for the ability to support growth of a cytokine-dependent cell line (Ba/F3) and for their capacity to induce TPO-stimulated tyrosine phosphorylation of specific signaling proteins. Point mutations of the five tyrosine residues in the cytoplasmic domain of the receptor were subsequently used to confirm our conclusions. From these studies we demonstrate that: (
i
) TPO-induced proliferation is moderately reduced by truncation of as many as 53 C-terminal amino acids of Mpl, including the sites of receptor tyrosine phosphorylation; (
ii
) truncation/mutation of residues 69–83 of the Mpl cytoplasmic domain enhances proliferative signaling, perhaps mediated by a decrease in receptor-driven cellular differentiation; (
iii
) Mpl can be phosphorylated at either Y112 or Y117 but not at the three proximal cytoplasmic tyrosine residues (Y8, Y29, and Y78); (
iv
) Y112 of Mpl is necessary for tyrosine phosphorylation of Shc and Shc-associated p145 (SHIP); and (
v
) unlike STAT3, STAT5 is partially phosphorylated in the absence of any tyrosine residues in the Mpl cytoplasmic domain. These studies identify subdomains of Mpl necessary for activation of several critical signaling pathways and point to two potentially novel mechanisms of TPO-induced signal transduction, an indirect pathway to STAT5 activation and a differentiation domain that acts by limiting proliferation.
α-Galactosidase A deficient mice: A model of Fabry disease
Authors: Ohshima Toshio , Murray Gary J. , Swaim William D. , Longenecker Glenn , Quirk Jane M. , Cardarelli Carol O. , Sugimoto Yoshikazu , Pastan Ira , Gottesman Michael M. , Brady Roscoe O. , Kulkarni Ashok B.
Published: July 2002

Fabry disease is an X-linked inherited metabolic disorder that is caused by a deficiency of α-galactosidase A (α-Gal A). Progressive deposition of neutral glycosphingolipids that have terminal α-linked galactosyl moieties in vascular endothelial cells causes renal failure along with premature myocardial infarctions and strokes in patients with this condition. No specific treatment is available for patients with this disorder at this time. An animal model of this condition would be valuable for exploring therapeutic strategies for patients with Fabry disease. We report here the generation of
α-Gal A
deficient mice by gene targeting and an analysis of the resulting phenotype. The knockout mice display a complete lack of α-Gal A activity. The mice, however, appeared clinically normal at 10 weeks of age. Ultrastructural analysis revealed concentric lamellar inclusions in the kidneys, and confocal microscopy using a fluorescent-labeled lectin specific for α-
d
-galactosyl residues showed accumulation of substrate in the kidneys as well as in cultured fibroblasts. Lipid analysis revealed a marked accumulation of ceramidetrihexoside in the liver and the kidneys. These findings indicate the similarity of the pathophysiological process in the mutant mice and in patients with Fabry disease. The deficiency of α-Gal A activity and the accumulation of material containing terminal α-galactosyl residues in cultured embryonic fibroblasts derived from
α-Gal A
(−/0) mice were corrected by transducing these cells with bicistronic multidrug resistance retroviruses containing human
α-Gal A
cDNA.
Chronic disability trends in elderly United States populations: 1982–1994
Authors: Manton Kenneth G. , Corder Larry , Stallard Eric
Published: July 2002

Statistically significant declines in chronic disability prevalence rates were observed in the elderly United States population between the 1982 and 1989 National Long Term Care Surveys (NLTCS). The 1994 NLTCS was used to investigate whether disability rate declines continued to 1994. The 1982, 1984, 1989, and 1994 NLTCS employ the same sample design and instrumentation so that trends in disability can be estimated with minimal sampling and measurement bias. Age (5-year categories from 65 to >95)-specific rates were calculated for the 1982 NLTCS and applied to United States Census Bureau estimates of the 1994 population to calculate chronic disability prevalence rates adjusted for aging in the United States population aged >65. The 1982 age standardized rates were compared with 1994 NLTCS estimates. The prevalence of disability estimated for 1994 (21.3%) was 3.6% lower than the 1982 age standardized rate (24.9%)—a highly significant reduction (t= −8.5;P≪ 0.0001). Of the 3.6 percentage point decline in prevalence, 1.7% occurred in the 5 years between 1989 and 1994—compared with the 1.9% decline in the 7 years between 1982 and 1989. Both declines are significant. Because of the shorter time period, the per year decline in disability prevalence from 1989 to 1994 was greater than that from 1982 to 1989. Given the higher acute and long-term care service needs of the disabled elderly population, Medicare, Medicaid, and private health expenditures may be dramatically lower than if declines had not occurred.
Calorie restriction reduces the incidence of myeloid leukemia induced by a single whole-body radiation in C3H/He mice
Authors: Yoshida Kazuko , Inoue Tohru , Nojima Kumie , Hirabayashi Yoko , Sado Toshihiko
Published: July 2002

Dietary restriction, especially caloric restriction, is a major modifier in experimental carcinogenesis and is known to decrease significantly the incidence of neoplasms. Gross and Dreyfuss [Gross, L. & Dreyfuss, Y. (1984)Proc. Natl. Acad. Sci. USA81, 7596–7598; Gross, L. & Dreyfuss, Y. (1986)Proc. Natl. Acad. Sci. USA83, 7928–7931] reported that a 36% restriction in caloric intake dramatically decreased the radiation-induced solid tumors and/or leukemias. Their protocol predominantly produced lymphatic neoplasms. It is of interest to observe the effect of caloric restriction on radiation-induced myeloid leukemia, because the disease was observed to have been increased in the survivors of the atomic bombs in Hiroshima and Nagasaki. The spontaneous incidence of myeloid leukemia in C3H/He male mice is 1%, and the incidence increased to 23.3% when 3 Gy of whole-body x-ray irradiation was given. However, the incidence of myeloid leukemia was found to be significantly decreased by caloric restriction; it was reduced to 7.9% and 10.7% when restriction was started before (6 weeks old) and after (10 weeks old) irradiation, respectively. In addition, the onset of the myeloid leukemia in both restricted groups was prolonged to a greater extent as compared with the control diet group. Caloric restriction demonstrated a significant prolongation of the life span in the groups on a restricted diet after having been exposed to irradiation, either before or after dietary restriction, in comparison with mice that were only irradiated.
p110δ, a novel phosphoinositide 3-kinase in leukocytes
Authors: Vanhaesebroeck Bart , Welham Melanie J. , Kotani Kei , Stein Rob , Warne Patricia H. , Zvelebil Markéta J. , Higashi Kyoichiro , Volinia Stefano , Downward Julian , Waterfield Michael D.
Published: July 2002

Phosphoinositide 3-kinases (PI3Ks) are a family of lipid kinases that have been implicated in signal transduction through tyrosine kinase- and heterotrimeric G-protein-linked receptors. We report herein the cloning and characterization of p110δ, a novel class I PI3K. Like p110α and p110β, other class I PI3Ks, p110δ displays a broad phosphoinositide lipid substrate specificity and interacts with SH2/SH3 domain-containing p85 adaptor proteins and with GTP-bound Ras. In contrast to the widely distributed p110α and β, p110δ is exclusively found in leukocytes. In these cells, p110α and δ both associate with the p85α and β adaptor subunits and are similarly recruited to activated signaling complexes after treatment with the cytokines interleukin 3 and 4 and stem cell factor. Thus, these class I PI3Ks appear not to be distinguishable at the level of p85 adaptor selection or recruitment to activated receptor complexes. However, distinct biochemical and structural features of p110δ suggest divergent functional/regulatory capacities for this PI3K. Unlike p110α, p110δ does not phosphorylate p85 but instead harbors an intrinsic autophosphorylation capacity. In addition, the p110δ catalytic domain contains unique potential protein–protein interaction modules such as a Pro-rich region and a basic-region leucine-zipper (bZIP)-like domain. Possible selective functions of p110δ in white blood cells are discussed.
Independent, specialized invasions of ectomycorrhizal mutualism by two nonphotosynthetic orchids
Authors: Taylor D. Lee , Bruns Thomas D.
Published: July 2002

We have investigated the mycorrhizal associations of two nonphotosynthetic orchids from distant tribes within the Orchidaceae. The two orchids were found to associate exclusively with two distinct clades of ectomycorrhizal basidiomycetous fungi over wide geographic ranges. Yet both orchids retained the internal mycorrhizal structure typical of photosynthetic orchids that do not associate with ectomycorrhizal fungi. Restriction fragment length polymorphism and sequence analysis of two ribosomal regions along with fungal isolation provided congruent, independent evidence for the identities of the fungal symbionts. All 14 fungal entities that were associated with the orchid
Cephalanthera austinae
belonged to a clade within the Thelephoraceae, and all 18 fungal entities that were associated with the orchid
Corallorhiza maculata
fell within the Russulaceae. Restriction fragment length polymorphism and single-strand conformational polymorphism analysis of ectomycorrhizal tree roots collected adjacent to
Cephalanthera
showed that (
i
) the fungi associated internally with
Cephalanthera
also form typical external ectomycorrhizae and that (
ii
) ectomycorrhizae formed by other Basidiomycetes were abundant where the orchid grows but these fungi did not associate with the orchid. This is the first proof of ectomycorrhizal epiparasitism in nature by an orchid. We argue that these orchids are cheaters because they do not provide fixed carbon to associated fungi. This view suggests that mycorrhizae, like other ancient mutualisms, are susceptible to cheating. The extreme specificity in these orchids relative to other ectomycorrhizal plants agrees with trends seen in more conventional parasites.
Locomotion: Dealing with friction
Authors: Radhakrishnan V.
Published: July 2002

To move on land, in water, or in the air, even at constant speed and at the same level, always requires an expenditure of energy. The resistance to motion that has to be overcome is of many different kinds depending on size, speed, and the characteristics of the medium, and is a fascinating subject in itself. Even more interesting are nature’s stratagems and solutions toward minimizing the effort involved in the locomotion of different types of living creatures, and humans’ imitations and inventions in an attempt to do at least as well.
Chromosomal instability in the descendants of unirradiated surviving cells after α-particle irradiation
Authors: Lorimore S. A. , Kadhim M. A. , Pocock D. A. , Papworth D. , Stevens D. L. , Goodhead D. T. , Wright E. G.
Published: July 2002

We have demonstrated chromosomal instability in the clonal descendants of hemopoietic stem cells after irradiating murine bone marrow with α-particles. However, because cells that are irradiated by α-particles are defined by a Poisson distribution of individual particle traversals, there is an inevitable proportion of unirradiated cells in the surviving population. The calculated expected proportions of irradiated and nonirradiated cells indicate that the number of clonogenic cells transmitting chromosomal instability is greater than the number expected to be hit and survive. To investigate further this discrepancy, we studied the effects of interposing a grid between the cells and the α-particle source so that the surviving population consists predominantly of untraversed stem cells. Comparison with the same irradiation conditions without the grid reveals that the same level of instability is induced. The data confirm that α-particles induce chromosomal instability but instability is demonstrated in the progeny of nonirradiated stem cells and must be due to unexpected interactions between irradiated and nonirradiated cells. This untargeted effect has important implications for mechanistic studies of radiation action and for assessment of radiation risk.
Structural basis for chemical inhibition of human blood coagulation factor Xa
Authors: Kamata Kenji , Kawamoto Hiroshi , Honma Teruki , Iwama Toshiharu , Kim Sung-Hou
Published: July 2002

Factor Xa, the converting enzyme of prothrombin to thrombin, has emerged as an alternative (to thrombin) target for drug discovery for thromboembolic diseases. An inhibitor has been synthesized and the crystal structure of the complex between Des[1–44] factor Xa and the inhibitor has been determined by crystallographic methods in two different crystal forms to 2.3- and 2.4-Å resolution. The racemic mixture of inhibitor FX-2212, (2
RS
)-(3′-amidino-3-biphenylyl)-5-(4-pyridylamino)pentanoic acid, inhibits factor Xa activity by 50% at 272 nM
in vitro
. The
S
-isomer of FX-2212 (FX-2212a) was found to bind to the active site of factor Xa in both crystal forms. The biphenylamidine of FX-2212a occupies the S1-pocket, and the pyridine ring makes hydrophobic interactions with the factor Xa aryl-binding site. Several water molecules meditate inhibitor binding to residues in the active site. In contrast to the earlier crystal structures of factor Xa, such as those of apo-Des[1–45] factor Xa and Des[1–44] factor Xa in complex with a naphthyl inhibitor DX-9065a, two epidermal growth factor-like domains of factor Xa are well ordered in both our crystal forms as well as the region between the two domains, which recently was found to be the binding site of the effector cell protease receptor-1. This structure provides a basis for designing next generation inhibitors of factor Xa.