bluuebunny/crossref_metadata_2025 huggingface dataset:
Microtubule self-organization is gravity-dependent
Authors: Papaseit Cyril , Pochon Nathalie , Tabony James
Published: July 2002

Although weightlessness is known to affect living cells, the manner by which this occurs is unknown. Some reaction-diffusion processes have been theoretically predicted as being gravity-dependent. Microtubules, a major constituent of the cellular cytoskeleton, self-organize
in vitro
by way of reaction-diffusion processes. To investigate how self-organization depends on gravity, microtubules were assembled under low gravity conditions produced during space flight. Contrary to the samples formed on an in-flight 1 ×
g
centrifuge, the samples prepared in microgravity showed almost no self-organization and were locally disordered.
Hsp70 and Hsp40 chaperones can inhibit self-assembly of polyglutamine proteins into amyloid-like fibrils
Authors: Muchowski Paul J. , Schaffar Gregor , Sittler Annie , Wanker Erich E. , Hayer-Hartl Manajit K. , Hartl F. Ulrich
Published: July 2002

The deposition of protein aggregates in neurons is a hallmark of neurodegenerative diseases caused by polyglutamine (polyQ) proteins. We analyzed the effects of the heat shock protein (Hsp) 70 chaperone system on the aggregation of fragments of huntingtin (htt) with expanded polyQ tracts.
In vitro
, Hsp70 and its cochaperone Hsp40 suppressed the assembly of htt into detergent-insoluble amyloid-like fibrils in an ATP-dependent manner and caused the formation of amorphous, detergent-soluble aggregates. The chaperones were most active in preventing fibrillization when added during the lag phase of the polymerization reaction. Similarly, coexpression of Hsp70 or Hsp40 with htt in yeast inhibited the formation of large, detergent-insoluble polyQ aggregates, resulting in the accumulation of detergent-soluble inclusions. Thus, the recently established potency of Hsp70 and Hsp40 to repress polyQ-induced neurodegeneration may be based on the ability of these chaperones to shield toxic forms of polyQ proteins and to direct them into nontoxic aggregates.
A novel mammalian receptor for the evolutionarily conserved type II GnRH
Authors: Millar Robert , Lowe Steven , Conklin Darrell , Pawson Adam , Maudsley Stuart , Troskie Brigitte , Ott Thomas , Millar Michael , Lincoln Gerald , Sellar Robin , Faurholm Bjarne , Scobie Graeme , Kuestner Rolf , Terasawa Ei , Katz Arieh
Published: July 2002

Mammalian gonadotropin-releasing hormone (GnRH I:
pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH
2
) stimulates
pituitary gonadotropin secretion, which in turn stimulates the gonads.
Whereas a hypothalamic form of GnRH of variable structure (designated
type I) had been shown to regulate reproduction through a cognate type
I receptor, it has recently become evident that most vertebrates have
one or two other forms of GnRH. One of these, designated type II GnRH
(GnRH II: pGlu-His-Ser-His-Gly-Trp-Tyr-Pro-Gly-NH
2
), is
conserved from fish to man and is widely distributed in the brain,
suggesting important neuromodulatory functions such as regulating
K
+
channels and stimulating sexual arousal. We now report
the cloning of a type II GnRH receptor from marmoset cDNA. The receptor
has only 41% identity with the type I receptor and, unlike the type I
receptor, has a carboxyl-terminal tail. The receptor is highly
selective for GnRH II. As with the type I receptor, it couples to
G
α
q/11
and also activates extracellular
signal-regulated kinase (ERK1/2) but differs in activating p38
mitogen activated protein (MAP) kinase. The type II receptor is more
widely distributed than the type I receptor and is expressed throughout
the brain, including areas associated with sexual arousal, and in
diverse non-neural and reproductive tissues, suggesting a variety of
functions. Surprisingly, the type II receptor is expressed in the
majority of gonadotropes. The presence of two GnRH receptors in
gonadotropes, together with the differences in their signaling,
suggests different roles in gonadotrope functioning.
Deficiency of human BRCA2 leads to impaired homologous recombination but maintains normal nonhomologous end joining
Authors: Xia Fen , Taghian Danielle G. , DeFrank Jeffrey S. , Zeng Zhao-Chong , Willers Henning , Iliakis George , Powell Simon N.
Published: July 2002

Carriers of BRCA2 germline mutations are at high risk to develop early-onset breast cancer. The underlying mechanisms of how BRCA2 inactivation predisposes to malignant transformation have not been established. Here, we provide direct functional evidence that human BRCA2 promotes homologous recombination (HR), which comprises one major pathway of DNA double-strand break repair. We found that up-regulated HR after transfection of wild-type (wt) BRCA2 into a human tumor line with mutant BRCA2 was linked to increased radioresistance. In addition, BRCA2-mediated enhancement of HR depended on the interaction with Rad51. In contrast to the tumor suppressor BRCA1, which is involved in multiple DNA repair pathways, BRCA2 status had no impact on the other principal double-strand break repair pathway, nonhomologous end joining. Thus, there exists a specific regulation of HR by BRCA2, which may function to maintain genomic integrity and suppress tumor development in proliferating cells.
M cell-targeted DNA vaccination
Authors: Wu Yunpeng , Wang Xinhai , Csencsits Keri L. , Haddad Asmahan , Walters Nancy , Pascual David W.
Published: July 2002

DNA immunization, although attractive, is poor for inducing mucosal immunity, thus limiting its protective value against most infectious agents. To surmount this shortcoming, we devised a method for mucosal transgene vaccination by using an M cell ligand to direct the DNA vaccine to mucosal inductive tissues and the respiratory epithelium. This ligand, reovirus protein σ1, when conjugated to polylysine (PL), can bind the apical surface of M cells from nasal-associated lymphoid tissues. Intranasal immunizations with protein σ1-PL-DNA complexes produced antigen-specific serum IgG and prolonged mucosal IgA, as well as enhanced cell-mediated immunity, made evident by elevated pulmonary cytotoxic T lymphocyte responses. Therefore, targeted transgene vaccination represents an approach for enabling DNA vaccination of the mucosa.
Targeted expression of heme oxygenase-1 prevents the pulmonary inflammatory and vascular responses to hypoxia
Authors: Minamino Tohru , Christou Helen , Hsieh Chung-Ming , Liu Yuxiang , Dhawan Vijender , Abraham Nader G. , Perrella Mark A. , Mitsialis S. Alex , Kourembanas Stella
Published: July 2002

Chronic hypoxia causes pulmonary hypertension with smooth muscle cell proliferation and matrix deposition in the wall of the pulmonary arterioles. We demonstrate here that hypoxia also induces a pronounced inflammation in the lung before the structural changes of the vessel wall. The proinflammatory action of hypoxia is mediated by the induction of distinct cytokines and chemokines and is independent of tumor necrosis factor-α signaling. We have previously proposed a crucial role for heme oxygenase-1 (HO-1) in protecting cardiomyocytes from hypoxic stress, and potent anti-inflammatory properties of HO-1 have been reported in models of tissue injury. We thus established transgenic mice that constitutively express HO-1 in the lung and exposed them to chronic hypoxia. HO-1 transgenic mice were protected from the development of both pulmonary inflammation as well as hypertension and vessel wall hypertrophy induced by hypoxia. Significantly, the hypoxic induction of proinflammatory cytokines and chemokines was suppressed in HO-1 transgenic mice. Our findings suggest an important protective function of enzymatic products of HO-1 activity as inhibitors of hypoxia-induced vasoconstrictive and proinflammatory pathways.
Differential effects of p19Arfand p16Ink4aloss on senescence of murine bone marrow-derived preB cells and macrophages
Authors: Randle David H. , Zindy Frederique , Sherr Charles J. , Roussel Martine F.
Published: July 2002

Establishment of cell lines from primary mouse embryo fibroblasts depends on loss of either theArftumor suppressor or its downstream target, the p53 transcription factor. Mouse p19Arfis encoded by theInk4a-Arflocus, which also specifies a second tumor suppressor protein, the cyclin D-dependent kinase inhibitor p16Ink4a. We surveyed bone marrow-derived cells from wild-type,Ink4a-Arf-null, orArf-null mice for their ability to bypass senescence during continuous passage in culture. Unlike preB cells from wild-type mice, those from mice lackingArfalone could be propagated indefinitely when placed onto stromal feeder layers engineered to produce IL-7. The preB cell lines remained diploid and IL-7-dependent and continued to express elevated levels of p16Ink4a. By contrast,Arf-null bone marrow-derived macrophages that depend on colony-stimulating factor-1 for proliferation and survival in culture initially grew at a slow rate but gave rise to rapidly and continuously growing, but still growth factor-dependent, variants that ceased to express p16Ink4a. Wild-type bone marrow-derived macrophages initially expressed both p16Ink4aand p19Arfbut exhibited an extended life span when p16Ink4aexpression was extinguished. In all cases, gene silencing was accompanied by methylation of theInk4apromoter. Therefore, whereasArfloss alone appears to be the major determinant of establishment of murine fibroblast and preB cell lines in culture, p16Ink4aprovides an effective barrier to immortalization of bone marrow-derived macrophages.
Dehydroascorbic acid, a blood–brain barrier transportable form of vitamin C, mediates potent cerebroprotection in experimental stroke
Authors: Huang Judy , Agus David B. , Winfree Christopher J. , Kiss Szilard , Mack William J. , McTaggart Ryan A. , Choudhri Tanvir F. , Kim Louis J , Mocco J , Pinsky David J. , Fox William D. , Israel Robert J. , Boyd Thomas A. , Golde David W. , Connolly E. Sander
Published: July 2002

Neuronal injury in ischemic stroke is partly mediated by cytotoxic
reactive oxygen species. Although the antioxidant ascorbic acid (AA) or
vitamin C does not penetrate the blood–brain barrier (BBB),
its oxidized form, dehydroascorbic acid (DHA), enters the brain by
means of facilitative transport. We hypothesized that i.v. DHA would
improve outcome after stroke because of its ability to cross the BBB
and augment brain antioxidant levels. Reversible or permanent focal
cerebral ischemia was created by intraluminal middle cerebral artery
occlusion in mice treated with vehicle, AA, or DHA (40, 250, or 500
mg/kg), either before or after ischemia. Given before ischemia, DHA
caused dose-dependent increases in postreperfusion cerebral blood flow,
with reductions in neurological deficit and mortality. In reperfused
cerebral ischemia, mean infarct volume was reduced from 53% and 59%
in vehicle- and AA-treated animals, respectively, to 15% in 250
mg/kg DHA-treated animals (
P
< 0.05). Similar
significant reductions occurred in nonreperfused cerebral ischemia.
Delayed postischemic DHA administration after 15 min or 3 h also
mediated improved outcomes. DHA (250 mg/kg or 500 mg/kg)
administered at 3 h postischemia reduced infarct volume by 6- to
9-fold, to only 5% with the highest DHA dose (
P
<
0.05). In contrast, AA had no effect on infarct volumes, mortality, or
neurological deficits. No differences in the incidence of intracerebral
hemorrhage occurred. Unlike exogenous AA, DHA confers
in
vivo
, dose-dependent neuroprotection in reperfused and
nonreperfused cerebral ischemia at clinically relevant times. As a
naturally occurring interconvertible form of AA with BBB permeability,
DHA represents a promising pharmacological therapy for stroke based on
its effects in this model of cerebral ischemia.
Pronounced climatic variations in Alaska during the last two millennia
Authors: Hu Feng Sheng , Ito Emi , Brown Thomas A. , Curry B. Brandon , Engstrom Daniel R.
Published: July 2002

Paired oxygen-isotopic analyses of abiotic carbonate and benthic-ostracode shells from lake sediments provide a continuous quantitative record of growing-season temperature for the past 2000 years in the northwestern foothills of the Alaska Range. This record reveals three time intervals of comparable warmth:
anno Domini
(A.D.) 0–300, 850-1200, and post-1800, the latter two of which correspond to the Medieval Climatic Anomaly and climatic amelioration after the end of the Little Ice Age. The Little Ice Age culminated at A.D. 1700, when the climate was ≈1.7°C colder than at present. A marked climatic cooling also occurred around A.D. 600, coinciding with extensive glacial advances in Alaska. Comparisons of this temperature record with ostracode trace-element ratios (Mg/Ca, Sr/Ca) further suggest that colder periods were wetter and vice versa during the past 2000 years.
Rhesus monkey placental transgene expression after lentiviral gene transfer into preimplantation embryos
Authors: Wolfgang M. J. , Eisele S. G. , Browne M. A. , Schotzko M. L. , Garthwaite M. A. , Durning M. , Ramezani A. , Hawley R. G. , Thomson J. A. , Golos T. G.
Published: July 2002

Transgenic mice have provided invaluable information about gene function and regulation. However, because of marked differences between rodents and primates, some areas of human biology such as early embryonic development, aging, and maternal–fetal interactions would be best studied in a nonhuman primate model. Here, we report that gene transfer into rhesus monkey (Macaca mulatta) preimplantation embryos gives rise to transgenic placentas that express a reporter transgene (eGFP). Blastocysts resulting from culture ofin vitrofertilized ova were transduced with a self-inactivating lentiviral vector and transferred into recipient females. One twin and one singleton pregnancy were produced from a single stimulation cycle, and one live rhesus monkey was born from each pregnancy. Placentas from all conceptuses showed expression of the transgene as detected by reverse transcription–PCR, ribonuclease protection assay, direct epifluorescence, immunohistochemistry, and Western blot analysis. Integration in somatic tissues of the offspring was not detected. A maternal immune response to the xenogeneic placental antigen was shown by the presence of anti-GFP antibodies in peripheral blood of the recipient females by day 99 of gestation (term = 165 days). These results demonstrate that transgene expression during gestation is compatible with successful pregnancy in nonhuman primates and provides an approach that could be broadly applicable to the development of novel models for primate biomedical research.
Human DNA replication initiation factors, ORC and MCM, associate with
oriP
of Epstein–Barr virus
Authors: Chaudhuri Biswendu , Xu Hongzhan , Todorov Ivan , Dutta Anindya , Yates John L.
Published: July 2002

The 165-kb chromosome of Epstein–Barr virus (EBV) is replicated by cellular enzymes only once per cell cycle in human cells that are latently infected. Here, we report that the human origin recognition complex, ORC, can be detected in association with an EBV replication origin,
oriP
, in cells by using antibodies against three different subunits of human ORC to precipitate crosslinked chromatin. Mcm2, a subunit of the MCM replication licensing complex, was found to associate with
oriP
during G
1
and to dissociate from it during S phase. The detection of ORC and Mcm2 at
oriP
was shown to require the presence of the 120-bp replicator of
oriP
. Licensing and initiation of replication at
oriP
of EBV thus seem to be mediated by ORC. This is an example of a virus apparently using ORC and associated factors for the propagation of its genome.
Plant gene expression response to
Agrobacterium tumefaciens
Authors: Ditt Renata F. , Nester Eugene W. , Comai Luca
Published: July 2002

To elucidate the nature of plant response to infection and transformation by
Agrobacterium tumefaciens
, we compared the cDNA-amplified fragment length polymorphism (AFLP) pattern of
Agrobacterium
- and mock-inoculated
Ageratum conyzoides
plant cell cultures. From 16,000 cDNA fragments analyzed, 251 (1.6%) were differentially regulated (0.5% down-regulated) 48 h after cocultivation with
Agrobacterium
. From 75 strongly regulated fragments, 56 were already regulated 24 h after cocultivation. Sequence similarities were obtained for 20 of these fragments, and reverse transcription–PCR analysis was carried out with seven to confirm their cDNA-AFLP differential pattern. Their sequence similarities suggest a role for these genes in signal perception, transduction, and plant defense. Reverse transcription–PCR analysis indicated that four genes involved in defense response are regulated in a similar manner by nonpathogenic bacteria, whereas one gene putatively involved in signal transduction appeared to respond more strongly to
Agrobacterium
. A nodulin-like gene was regulated only by
Agrobacterium
. These results demonstrate a rapid plant cell response to
Agrobacterium
infection, which overlaps a general response to bacteria but also has
Agrobacterium
-specific features.
In vitro
reconstitution of the myxochelin biosynthetic machinery of
Stigmatella aurantiaca
Sg a15: Biochemical characterization of a reductive release mechanism from nonribosomal peptide synthetases
Authors: Gaitatzis Nikolaos , Kunze Brigitte , Müller Rolf
Published: July 2002

Microorganisms produce iron-chelating compounds to sequester the
iron essential for growth from the environment. Many of these compounds
are biosynthesized by nonribosomal peptide synthetases, some in
cooperation with polyketide synthases. Myxochelins are produced by the
myxobacterium
Stigmatella aurantiaca
Sg a15, and the
corresponding gene cluster was cloned recently. We have undertaken to
express heterologously the myxochelin biosynthetic machinery in
Escherichia coli
. To activate the involved proteins
posttranslationally, they were coexpressed with the
phosphopantetheinyltransferase MtaA from the myxothiazol biosynthetic
gene cluster. Phosphopantetheinylation of the carrier proteins could be
verified by protein mass analysis. Six active domains in proteins MxcE,
MxcF, and MxcG are capable of assembling myxochelin from ATP, NAD(P)H,
lysine, and 2,3-dihydroxybenzoic acid
in vitro
. This
fact demonstrates that the condensation domain of MxcG performs two
condensation reactions, creating the aryl-capped α-amide and the
aryl-capped γ-amide of the molecule. A previously unknown type of
reductive release is performed by the reduction domain of MxcG, which
alternatively uses NADPH and NADH to set free the peptidyl-carrier
protein-bound thioester as an aldehyde and further reduces it to the
alcohol structure that can be found in myxochelin A. This type of
reductive release seems to be a general mechanism in polyketide and
nonribosomal peptide biosynthesis, because several systems with
C-terminal similarity to the reductase domain of MxcG can be found in
the databases. Alternatively, the aldehyde can be transaminated, giving
rise to a terminal amine.
Purification and characterization of human DNA damage checkpoint Rad complexes
Authors: Lindsey-Boltz Laura A. , Bermudez Vladimir P. , Hurwitz Jerard , Sancar Aziz
Published: July 2002

Checkpoint Rad proteins function early in the DNA damage
checkpoint signaling cascade to arrest cell cycle progression in
response to DNA damage. This checkpoint ensures the transmission of an
intact genetic complement to daughter cells. To learn about the damage
sensor function of the human checkpoint Rad proteins, we purified a
heteropentameric complex composed of
hRad17-RFCp36-RFCp37-RFCp38-RFCp40 (hRad17-RFC) and a heterotrimeric
complex composed of hRad9-hHus1-hRad1 (checkpoint 9-1-1 complex).
hRad17-RFC binds to DNA, with a preference for primed DNA and possesses
weak ATPase activity that is stimulated by primed DNA and
single-stranded DNA. hRad17-RFC forms a complex with the 9-1-1
heterotrimer reminiscent of the replication factor C/proliferating
cell nuclear antigen clamp loader/sliding clamp complex of the
replication machinery. These findings constitute biochemical support
for models regarding the roles of checkpoint Rads as damage sensors in
the DNA damage checkpoint response of human cells.
Self-organizing biochemical cycles
Authors: Orgel Leslie E.
Published: July 2002

I examine the plausibility of theories that postulate the
development of complex chemical organization without requiring the
replication of genetic polymers such as RNA. One conclusion is that
theories that involve the organization of complex, small-molecule
metabolic cycles such as the reductive citric acid cycle on mineral
surfaces make unreasonable assumptions about the catalytic properties
of minerals and the ability of minerals to organize sequences of
disparate reactions. Another conclusion is that data in the Beilstein
Handbook of Organic Chemistry
that have been claimed to
support the hypothesis that the reductive citric acid cycle originated
as a self-organized cycle can more plausibly be interpreted in a
different way.
Increased p53 mutation load in nontumorous human liver of Wilson disease and hemochromatosis: Oxyradical overload diseases
Authors: Hussain S. Perwez , Raja Kamran , Amstad Paul A. , Sawyer Mark , Trudel Laura J. , Wogan Gerald N. , Hofseth Lorne J. , Shields Peter G. , Billiar Timothy R. , Trautwein Christian , Höhler Thomas , Galle Peter R. , Phillips David H. , Markin Rodney , Marrogi Aizen J. , Harris Curtis C.
Published: July 2002

Hemochromatosis and Wilson disease (WD), characterized by the
excess hepatic deposition of iron and copper, respectively, produce
oxidative stress and increase the risk of liver cancer. Because the
frequency of p53 mutated alleles in nontumorous human tissue may be a
biomarker of oxyradical damage and identify individuals at increased
cancer risk, we have determined the frequency of p53 mutated alleles in
nontumorous liver tissue from WD and hemochromatosis patients. When
compared with the liver samples from normal controls, higher
frequencies of G:C to T:A transversions at codon 249
(
P
< 0.001) and C:G to A:T transversions and C:G
to T:A transitions at codon 250 (
P
< 0.001 and
P
< 0.005) were found in liver tissue from WD
cases, and a higher frequency of G:C to T:A transversions at codon 249
(
P
< 0.05) also was found in liver tissue from
hemochromatosis cases. Sixty percent of the WD and 28% of
hemochromatosis cases also showed a higher expression of inducible
nitric oxide synthase in the liver, which suggests nitric oxide as a
source of increased oxidative stress. A high level of etheno-DNA
adducts, formed from oxyradical-induced lipid peroxidation, in liver
from WD and hemochromatosis patients has been reported previously.
Therefore, we exposed a wild-type p53 TK-6 lymphoblastoid cell line to
4-hydroxynonenal, an unsaturated aldehyde involved in lipid
peroxidation, and observed an increase in G to T transversions at p53
codon 249 (AGG to AGT). These results are consistent with the
hypothesis that the generation of oxygen/nitrogen species and
unsaturated aldehydes from iron and copper overload in hemochromatosis
and WD causes mutations in the p53 tumor suppressor gene.
Cytotoxic T lymphocyte antigen-4 (CTLA-4) regulates the size, reactivity, and function of a primed pool of CD4+T cells
Authors: Kuhns Michael S. , Epshteyn Victoria , Sobel Raymond A. , Allison James P.
Published: July 2002

We examined how cytotoxic T lymphocyte antigen-4 (CTLA-4) regulates heterogeneous CD4+T cell responses by using experimental autoimmune encephalomyelitis (EAE), a CD4+T cell-mediated disease that is subject to regulation by CTLA-4. Disease incidence and severity were used as measures ofin vivoCD4+T cell responses. The frequency, cytokine production, and reactivity of primed T cells were determined from animals immunized with proteolipid protein (PLP)-139–151 (disease agonist), PLP-Q (disease antagonist), or both peptides, and treated with control or anti-CTLA-4 antibody to analyze the responding population. CTLA-4 blockade exacerbated disease in PLP-139–151-primed animals and overcame disease antagonism in coimmunized animals, but did not permit disease induction in PLP-Q-primed animals. Experimental autoimmune encephalomyelitis enhancement was associated with increased frequencies of cytokine-producing cells and increased ratios of IFN-γ to IL-4 secretors responsive to PLP-139–151. Priming with PLP-Q elicited IL-4 and IL-2, but not IFN-γ secretors cross-reactive with PLP-139–151. Strikingly, CTLA-4 blockade was found to decrease rather than increase the frequencies of cross-reactive IL-4 and IL-2 secretors. Thus, CTLA-4 engagement limits the size, but increases the breadth, of reactivity of a primed pool of CD4+T cells, consequently regulating its function.
Vigilin binding selectively inhibits cleavage of the vitellogenin mRNA 3′-untranslated region by the mRNA endonuclease polysomal ribonuclease 1
Authors: Cunningham Kristopher S. , Dodson Robin E. , Nagel Maria A. , Shapiro David J. , Schoenberg Daniel R.
Published: July 2002

In
Xenopus
, estrogen induces the stabilization of
vitellogenin mRNA and the destabilization of albumin mRNA. These
processes correlate with increased polysomal activity of a
sequence-selective mRNA endonuclease, PMR-1, and a hnRNP K
homology-domain RNA-binding protein, vigilin. Vigilin binds to a region
of the vitellogenin mRNA 3′-untranslated region (3′-UTR) implicated in
estrogen-mediated stabilization. The vigilin-binding site in the
vitellogenin B1 mRNA 3′-UTR contains two consensus PMR-1 cleavage
sites. The availability of purified PMR-1 and recombinant vigilin made
it possible to test the hypothesis that RNA-binding proteins interact
with cis-acting elements to stabilize target mRNAs by blocking cleavage
by site-specific mRNA endonucleases. Vigilin binds to the vitellogenin
mRNA 3′-UTR site with at least 30-fold higher affinity than it exhibits
for the albumin mRNA segment containing the mapped PMR-1 cleavage
sites. This differential binding affinity correlates with differential
in vitro
susceptibility of the protein–RNA complexes to
cleavage by PMR-1. Whereas recombinant vigilin has no detectable
protective effect on PMR-1 cleavage of albumin mRNA, it retards
in vitro
cleavage of the vitellogenin mRNA 3′-UTR by
purified PMR-1. The PMR-1 sites in the vitellogenin mRNA 3′-UTR are
functional because they are readily cleaved
in vitro
by
purified PMR-1. These results provide direct evidence for differential
susceptibility to endonuclease-mediated mRNA decay resulting from the
differential affinity of a RNA-binding protein for cis-acting stability
determinants.
Linking immune-mediated arterial inflammation and cholesterol-induced atherosclerosis in a transgenic mouse model
Authors: Ludewig Burkhard , Freigang Stefan , Jäggi Martin , Kurrer Michael O. , Pei Yao-Chang , Vlk Lenka , Odermatt Bernhard , Zinkernagel Rolf M. , Hengartner Hans
Published: July 2002

Arterial inflammatory responses are thought to be a significant
component of atherosclerotic disease. We describe here, using a
transgenic approach, the mutual perpetuation of immune-mediated
arterial inflammation and cholesterol-induced atherosclerosis. Mice
expressing the bacterial transgene β-galactosidase exclusively in
cardiomyocytes and in smooth muscle cells in lung arteries and the
aorta (SM-LacZ), and hypercholesterolemic apolipoprotein E-deficient
SM-LacZ mice (SM-LacZ/apoE
−/−
) developed myocarditis
and arteritis after immunization with dendritic cells presenting a
β-galactosidase-derived immunogenic peptide. Hypercholesterolemia
amplified acute arteritis and perpetuated chronic arterial inflammation
in SM-LacZ/apoE
−/−
mice, but had no major impact on
acute myocarditis or the subsequent development of dilated
cardiomyopathy. Conversely, arteritis significantly accelerated
cholesterol-induced atherosclerosis. Taken together, these data
demonstrate that the linkage of immune-mediated arteritis and
hypercholesterolemia favors initiation and maintenance of
atherosclerotic lesion formation. Therapeutic strategies to prevent or
disrupt such self-perpetuating vicious circles may be crucial for the
successful treatment of atherosclerosis.
Long-term changes in Serengeti-Mara wildebeest and land cover: Pastoralism, population, or policies?
Authors: Homewood K. , Lambin E. F. , Coast E. , Kariuki A. , Kikula I. , Kivelia J. , Said M. , Serneels S. , Thompson M.
Published: July 2002

Declines in habitat and wildlife in semiarid African savannas are widely reported and commonly attributed to agropastoral population growth, livestock impacts, and subsistence cultivation. However, extreme annual and shorter-term variability of rainfall, primary production, vegetation, and populations of grazers make directional trends and causal chains hard to establish in these ecosystems. Here two decades of changes in land cover and wildebeest in the Serengeti-Mara region of East Africa are analyzed in terms of potential drivers (rainfall, human and livestock population growth, socio-economic trends, land tenure, agricultural policies, and markets). The natural experiment research design controls for confounding variables, and our conceptual model and statistical approach integrate natural and social sciences data. The Kenyan part of the ecosystem shows rapid land-cover change and drastic decline for a wide range of wildlife species, but these changes are absent on the Tanzanian side. Temporal climate trends, human population density and growth rates, uptake of small-holder agriculture, and livestock population trends do not differ between the Kenyan and Tanzanian parts of the ecosystem and cannot account for observed changes. Differences in private versus state/communal land tenure, agricultural policy, and market conditions suggest, and spatial correlations confirm, that the major changes in land cover and dominant grazer species numbers are driven primarily by private landowners responding to market opportunities for mechanized agriculture, less by agropastoral population growth, cattle numbers, or small-holder land use.
A model for gene therapy of human hereditary lymphedema
Authors: Karkkainen Marika J. , Saaristo Anne , Jussila Lotta , Karila Kaisa A. , Lawrence Elizabeth C. , Pajusola Katri , Bueler Hansruedi , Eichmann Anne , Kauppinen Risto , Kettunen Mikko I. , Ylä-Herttuala Seppo , Finegold David N. , Ferrell Robert E. , Alitalo Kari
Published: July 2002

Primary human lymphedema (Milroy's disease), characterized by a chronic and disfiguring swelling of the extremities, is associated with heterozygous inactivating missense mutations of the gene encoding vascular endothelial growth factor C/D receptor (VEGFR-3). Here, we describe a mouse model and a possible treatment for primary lymphedema. Like the human patients, the lymphedema (Chy) mice have an inactivating
Vegfr3
mutation in their germ line, and swelling of the limbs because of hypoplastic cutaneous, but not visceral, lymphatic vessels. Neuropilin (NRP)-2 bound VEGF-C and was expressed in the visceral, but not in the cutaneous, lymphatic endothelia, suggesting that it may participate in the pathogenesis of lymphedema. By using virus-mediated VEGF-C gene therapy, we were able to generate functional lymphatic vessels in the lymphedema mice. Our results suggest that growth factor gene therapy is applicable to human lymphedema and provide a paradigm for other diseases associated with mutant receptors.
Tetrahymena
proteins p80 and p95 are not core telomerase components
Authors: Mason Douglas X. , Autexier Chantal , Greider Carol W.
Published: July 2002

Telomeres provide stability to eukaryotic chromosomes and consist of tandem DNA repeat sequences. Telomeric repeats are synthesized and maintained by a specialized reverse transcriptase, termed telomerase.
Tetrahymena thermophila
telomerase contains two essential components:
Tetrahymena
telomerase reverse transcriptase (tTERT), the catalytic protein component, and telomerase RNA that provides the template for telomere repeat synthesis. In addition to these two components, two proteins, p80 and p95, were previously found to copurify with telomerase activity and to interact with tTERT and telomerase RNA. To investigate the role of p80 and p95 in the telomerase enzyme, we tested the interaction of p80, p95, and tTERT in several different recombinant expression systems and in
Tetrahymena
extracts. Immunoprecipitation of recombinant proteins showed that although p80 and p95 associated with each other, they did not associate with tTERT. In
in vitro
transcription and translation lysates, tTERT was associated with telomerase activity, but p80 and p95 were not. p80 bound telomerase RNA as well as several other unrelated RNAs, suggesting p80 has a general affinity for RNA. Immunoprecipitations from
Tetrahymena
extracts also showed no evidence for an interaction between the core tTERT/telomerase RNA complex and the p80 and p95 proteins. These data suggest that p80 and p95 are not associated with the bulk of active telomerase in
Tetrahymena
.
Virus-encoded suppressor of posttranscriptional gene silencing targets a maintenance step in the silencing pathway
Authors: Llave Cesar , Kasschau Kristin D. , Carrington James C.
Published: July 2002

Certain plant viruses encode suppressors of posttranscriptional
gene silencing (PTGS), an adaptive antiviral defense response that
limits virus replication and spread. The tobacco etch potyvirus
protein, helper component-proteinase (HC-Pro), suppresses PTGS of
silenced transgenes. The effect of HC-Pro on different steps of the
silencing pathway was analyzed by using both transient
Agrobacterium tumefaciens
-based delivery and transgenic
systems. HC-Pro inactivated PTGS in plants containing a preexisting
silenced β-glucuronidase (GUS) transgene. PTGS in this system was
associated with both small RNA molecules (21–26 nt) corresponding to
the 3′ proximal region of the transcribed GUS sequence and cytosine
methylation of specific sites near the 3′ end of the GUS transgene.
Introduction of HC-Pro into these plants resulted in loss of PTGS, loss
of small RNAs, and partial loss of methylation. These results suggest
that HC-Pro targets a PTGS maintenance (as opposed to an initiation or
signaling) component at a point that affects accumulation of small RNAs
and methylation of genomic DNA.
Reactivation of proliferin gene expression is associated with increased angiogenesis in a cell culture model of fibrosarcoma tumor progression
Authors: Toft Daniel J. , Rosenberg Suzanne B. , Bergers Gabriele , Volpert Olga , Linzer Daniel I. H.
Published: July 2002

Proliferin (PLF) is an angiogenic placental hormone. We now report that PLF gene expression can also occur in a progressive fibrosarcoma mouse tumor cell model. PLF mRNA and protein are detectable at very low levels in cell lines derived from the mild noninvasive stage of tumor development. Expression is greatly augmented in cell lines from the aggressively invasive stage of development, a stage at which the tumor becomes highly angiogenic, and PLF expression remains high in cell lines from the end stage of fibrosarcoma. Activator protein 1 factors present at high levels in the more invasive stages of the tumor may in part allow for increased PLF expression, as cells from the mild stage in which c-jun and junB are stably expressed secrete levels of PLF comparable to that of the advanced stages. Secreted PLF protein is functionally important in tumor cell angiogenic activity, as demonstrated by the reduction of angiogenic activity in fibrosarcoma cell culture medium by immunodepletion of PLF. These results suggest that an extraembryonic genetic program, which has evolved to support fetal growth, may be reactivated in certain tumors and contribute to tumor growth.
A transcription factor regulatory circuit in differentiated pancreatic cells
Authors: Boj Sylvia F. , Párrizas Marcelina , Maestro Miguel A. , Ferrer Jorge
Published: July 2002

Mutations in the human genes encoding hepatocyte nuclear factors
(HNF) 1α, 1β, 4α, and IPF1(PDX1/IDX1/STF1) result in
pancreatic β cell dysfunction and diabetes mellitus. In
hepatocytes,
hnf4α
controls the transcription of
hnf1α
, suggesting that this same interaction may
operate in β cells and thus account for the common diabetic
phenotype. We show that, in pancreatic islet and exocrine cells,
hnf4α
expression unexpectedly depends on
hnf1α
. This effect is tissue-specific and mediated
through direct occupation by hnf1α of an alternate promoter located
45.6 kb from the previously characterized
hnf4α
promoter.
Hnf1α
also exerts direct control of
pancreatic-specific expression of
hnf4
γ and
hnf3γ
.
Hnf1α
dependence of
hnf4α
,
hnf4γ
,
hnf3γ
,
and two previously characterized distal targets (
glut2
and
pklr
) is established only after differentiated cells
arise during pancreatic embryonic development. These studies define an
unexpected hierarchical regulatory relationship between two genes
involved in human monogenic diabetes in the cells, which are relevant
to its pathophysiology. Furthermore, they indicate that
hnf1α
is an essential component of a transcription
factor circuit whose role may be to maintain differentiated functions
of pancreatic cells.
Transgenic expression of the coxsackie/adenovirus receptor enables adenoviral-mediated gene delivery in naïve T cells
Authors: Wan Yisong Y. , Leon Ronald P. , Marks Reinhard , Cham Candace M. , Schaack Jerome , Gajewski Thomas F. , DeGregori James
Published: July 2002

The inability to easily and efficiently introduce genes into primary T cells has hampered the investigation of the pathways controlling T cell fate. To enable adenoviral-mediated gene transfer into normal naïve T cells, transgenic (Tg) mice expressing the coxsackie/adenovirus receptor (CAR) in their T cell compartment were constructed. Whereas naïve T cells are resistant to adenoviral infection, Tg expression of CAR on T cells greatly facilitates adenoviral-mediated gene expressionex vivo,in vivo, and in differentiated T helper cells. Thus we have developed a technology for efficient gene delivery to naïve T cells. By using adenoviral vectors encoding specific inhibitors, we show that G1 cyclin-dependent kinase, NF-κB, and caspase activities are required for the proliferation of primary T cells. In addition, by expressing Bcl-xLprotein at a level that closely approximates mitogen-induced levels, we demonstrate that Bcl-xLexpression is sufficient to account for mitogen-mediated survival of primary T cells. Thus, adenoviral-mediated gene delivery to CAR Tg T cells should be useful for the analysis of many genes controlling T cell fate.
Transgenesis by lentiviral vectors: Lack of gene silencing in mammalian embryonic stem cells and preimplantation embryos
Authors: Pfeifer Alexander , Ikawa Masahito , Dayn Yelena , Verma Inder M.
Published: July 2002

The introduction of foreign genes into early mouse embryos and embryonic stem (ES) cells is invaluable for the analysis of gene function and regulation in the living animal. The use of vectors derived from retroviruses as gene transfer vehicles in this setting has had limited success because of silencing of transgene expression. Here, we show that vectors derived from lentiviruses, which are complex retroviruses, can efficiently deliver genes to murine ES cells and that transgene expression is stable during proliferation of undifferentiated ES cells. The transgene is expressed during differentiation of ES cells
in vitro
(embryoid bodies) and
in vivo
(teratomas). Transfer of lentivector-transduced ES cells into blastocysts resulted in chimeric animals that expressed the transgene in multiple tissues. Embryos derived from crossings of chimeric mice expressed the transgene, indicating successful germ-line transmission. Infection of murine preimplantation embryos at morula stage with lentiviral vectors resulted in stable transduction and expression of the transgene in mouse embryos and in newborn mice. Finally, human ES cells were transduced by lentiviral vectors and expressed the transgene over several passages. Thus, lentiviral vectors represent a significant improvement over oncoretroviral vectors used previously for gene transfer into murine ES cells and preimplantation embryos. Ability to transfer foreign genes into human ES cells has potential relevance for the development of gene and cell-based therapies.
Disruption of primary auditory cortex by synchronous auditory inputs during a critical period
Authors: Zhang Li I. , Bao Shaowen , Merzenich Michael M.
Published: July 2002

In the primary auditory cortex (AI), the development of tone frequency selectivity and tonotopic organization is influenced by patterns of neural activity. Introduction of synchronous inputs into the auditory pathway achieved by exposing rat pups to pulsed white noise at a moderate intensity during P9–P28 resulted in a disrupted tonotopicity and degraded frequency-response selectivity for neurons in the adult AI. The latter was manifested by broader-than-normal tuning curves, multipeaks, and discontinuous, tone-evoked responses within AI-receptive fields. These effects correlated with the severe impairment of normal, developmental sharpening, and refinement of receptive fields and tonotopicity. In addition, paradoxically weaker than normal temporal correlations between the discharges of nearby AI neurons were recorded in exposed rats. In contrast, noise exposure of rats older than P30 did not cause significant change of auditory cortical maps. Thus, patterned auditory inputs appear to play a crucial role in shaping neuronal processing/decoding circuits in the primary auditory cortex during a critical period.
Molecular cloning of mesothelin, a differentiation antigen present on mesothelium, mesotheliomas, and ovarian cancers.
Authors: Chang K , Pastan I
Published: July 2002

Monoclonal antibody MAb K1 recognizes a 40-kDa glycoprotein present on the surface of mesothelial cells, mesotheliomas, and ovarian cancers. We have used MAb K1 to isolate a 2138-bp cDNA that encodes this antigen. The cDNA has an 1884-bp open reading frame encoding a 69-kDa protein. When the cDNA was transfected into COS and NIH 3T3 cells, the antigen was found on the cell surface and could be released by treatment with phosphatidylinositol-specific phospholipase C. The 69-kDa precursor is processed to the 40-kDa form. The protein has been named mesothelin because it is made by mesothelial cells. Mesothelin may play a role in cellular adhesion.
Smoothness within ruggedness: the role of neutrality in adaptation.
Authors: Huynen M A , Stadler P F , Fontana W
Published: July 2002

RNA secondary structure folding algorithms predict the existence of connected networks of RNA sequences with identical structure. On such networks, evolving populations split into subpopulations, which diffuse independently in sequence space. This demands a distinction between two mutation thresholds: one at which genotypic information is lost and one at which phenotypic information is lost. In between, diffusion enables the search of vast areas in genotype space while still preserving the dominant phenotype. By this dynamic the success of phenotypic adaptation becomes much less sensitive to the initial conditions in genotype space.
Gain of chromosome 3q defines the transition from severe dysplasia to invasive carcinoma of the uterine cervix.
Authors: Heselmeyer K , Schröck E , du Manoir S , Blegen H , Shah K , Steinbeck R , Auer G , Ried T
Published: July 2002

We have chosen tumors of the uterine cervix as a model system to identify chromosomal aberrations that occur during carcinogenesis. A phenotype/genotype correlation was established in defined regions of archived, formalin-fixed, and hematoxylin/eosin-stained tissue sections that were dissected from normal cervical epithelium (n = 3), from mild (n = 4), moderate (n = 6), and severe dysplasias/carcinomas in situ (CIS) (n = 13), and from invasive carcinomas (n = 10) and investigated by comparative genomic hybridization. The same tissues were analyzed for DNA ploidy, proliferative activity, and the presence of human papillomavirus (HPV) sequences. The results show that an increase in proliferative activity and tetraploidization had occurred already in mildly dysplastic lesions. No recurrent chromosomal aberrations were observed in DNA extracted from normal epithelium or from mild and moderate dysplasias, indicating that the tetraploidization precedes the loss or gain of specific chromosomes. A gain of chromosome 3q became visible in one of the severe dysplasias/CIS. Notably, chromosome 3q was overrepresented in 90% of the carcinomas and was also found to have undergone a high-level copy-number increase (amplification). We therefore conclude that the gain of chromosome 3q that occurs in HPV16-infected, aneuploid cells represents a pivotal genetic aberration at the transition from severe dysplasia/CIS to invasive cervical carcinoma.
The presence of codon-anticodon pairs in the acceptor stem of tRNAs.
Authors: Rodin S , Rodin A , Ohno S
Published: July 2002

A total of 1268 available (excluding mitochondrial) tRNA sequences was used to reconstruct the common consensus image of their acceptor domains. Its structure appeared as a 11-bp-long double-stranded palindrome with complementary triplets in the center, each flanked by the 3'-ACCD and NGGU-5' motifs on each strand (D, base determinator). The palindrome readily extends up to the modern tRNA-like cloverleaf passing through an intermediate hairpin having in the center the single-stranded triplet, in supplement to its double-stranded precursor. The latter might represent an original anticodon-codon pair mapped at 1-2-3 positions of the present-day tRNA acceptors. This conclusion is supported by the striking correlation: in pairs of consensus tRNAs with complementary anticodons, their bases at the 2nd position of the acceptor stem were also complementary. Accordingly, inverse complementarity was also evident at the 71st position of the acceptor stem. With a single exception (tRNA(Phe)-tRNA(Glu) pair), the parallelism is especially impressive for the pairs of tRNAs recognized by aminoacyl-tRNA synthetases (aaRS) from the opposite classes. The above complementarity still doubly presented at the key central position of real single-stranded anticodons and their hypothetical double-stranded precursors is consistent with our previous data pointing to the double-strand use of ancient RNAs in the origin of the main actors in translation- tRNAs with complementary anticodons and the two classes of aaRS.
RIG-E, a human homolog of the murine Ly-6 family, is induced by retinoic acid during the differentiation of acute promyelocytic leukemia cell.
Authors: Mao M , Yu M , Tong J H , Ye J , Zhu J , Huang Q H , Fu G , Yu L , Zhao S Y , Waxman S , Lanotte M , Wang Z Y , Tan J Z , Chan S J , Chen Z
Published: July 2002

In vivo all-trans-retinoic acid (ATRA), a differentiation inducer, is capable of causing clinical remission in about 90% of patients with acute promyelocytic leukemia (APL). The molecular basis for the differentiation of APL cells after treatment with ATRA remains obscure and may involve genes other than the known retinoid nuclear transcription factors. We report here the ATRA-induced gene expression in a cell line (NB4) derived from a patient with APL. By differential display-PCR, we isolated and characterized a novel gene (RIG-E) whose expression is up-regulated by ATRA. The gene is 4.0 kb long, consisting of four exons and three introns, and is localized on human chromosome region 8q24. The deduced amino acid sequence predicts a cell surface protein containing 20 amino acids at the N-terminal end corresponding to a signal peptide and an extracellular sequence containing 111 amino acids. The RIG-E coded protein shares some homology with CD59 and with a number of growth factor receptors. It shares high sequence homology with the murine LY-6 multigene family, whose members are small cysteine-rich proteins differentially expressed in several hematopoietic cell lines and appear to function in signal transduction. It seems that so far RIG-E is the closest human homolog of the LY-6 family. Expression of RIG-E is not restricted to myeloid differentiation, because it is also present in thymocytes and in a number of other tissues at different levels.