bluuebunny/crossref_metadata_2025 huggingface dataset:
Oscillatory brain states and learning: Impact of hippocampal theta-contingent training
Authors: Seager Matthew A. , Johnson Lynn D. , Chabot Elizabeth S. , Asaka Yukiko , Berry Stephen D.
Published: July 2002

Eyeblink classical conditioning is a relatively simple form of associative learning that has become an invaluable tool in our understanding of the neural mechanisms of learning. When studying rabbits in this paradigm, we observed a dramatic modification of learning rate by conducting training during episodes of either hippocampal theta or hippocampal non-theta activity as determined by on-line slow-wave spectral analysis. Specifically, if animals were given trials only when a computer analysis verified a predominance of slow-wave oscillations at theta frequencies (3–8 Hz), they learned in half as many trials as animals trained during non-theta hippocampal activity (58 vs. 115). This finding provides important evidence from awake, behaving animals that supports recent advances in our knowledge of (
i
) brain sites and neurobiological mechanisms of learning and memory, specifically hippocampus and theta oscillations, (
ii
) the biological plausibility of current models of hippocampal function that posit important roles for oscillatory potentials, and (
iii
) the design of interfaces between biological and cybernetic (electronic) systems that can optimize cognitive processes and performance.
A dynamic shift of VEGF isoforms with a transient and selective progesterone-induced expression of VEGF189regulates angiogenesis and vascular permeability in human uterus
Authors: Ancelin Magali , Buteau-Lozano Hélène , Meduri Geri , Osborne-Pellegrin Mary , Sordello Sylvie , Plouët Jean , Perrot-Applanat Martine
Published: July 2002

A key mechanism underlying physiological angiogenesis of the human endometrium is its ability to regenerate the vascular capillary network and to perform vascular remodeling (i.e., development of spiral arteries). Vascular endothelial growth factor (VEGF) is associated with angiogenesis and capillary permeability in this tissue. VEGF is expressed as several spliced variants, its main human isoforms contain 121 and 165 aa; 17β-estradiol (E2) increases endometrial VEGF, possibly in all isoforms. Here we show that progesterone (P) selectively increases the expression of the VEGF189(V189) isoform in the human uterus. V189is identified in the conditioned medium of stromal cells treated with E2+ P; its presence in thisin vitromodel of decidual stromal cells is detected after 6–8 days, using ELISA, and after 8–10 days, using Western blot analysis with different antibodies, including one specific for V189. The secretion pattern of V189parallels that of the decidual protein IGFBP-1. V189is secreted as a native isoform, as compared with the migration of recombinant V189by SDS/PAGE.In situhybridization and immunocytochemistry,performed on the same biopsies, suggest that decidual cells express V189during the mid-late secretory phase of the menstrual cycle and early gestation. Finally, using anin vivopermeability assay, we show that native V189increases capillary permeability. These observations demonstrate that P regulates V189expression in decidual cells, which could have important implications for understanding uterine vascular remodeling and implantation, and may be relevant in a range of disease states such as edema and irregular bleeding.
Linking osteopetrosis and pycnodysostosis: Regulation of cathepsin K expression by the microphthalmia transcription factor family
Authors: Motyckova G. , Weilbaecher K. N. , Horstmann M. , Rieman D. J. , Fisher D. Z. , Fisher D. E.
Published: July 2002

Various genetic conditions produce dysfunctional osteoclasts
resulting in osteopetrosis or osteosclerosis. These include human
pycnodysostosis, an autosomal recessive syndrome caused by cathepsin K
mutation, cathepsin K-deficient mice, and
mitf
mutant
rodent strains. Cathepsin K is a highly expressed cysteine protease in
osteoclasts that plays an essential role in the degradation of protein
components of bone matrix. Cathepsin K also is expressed in a
significant fraction of human breast cancers where it could contribute
to tumor invasiveness. Mitf is a member of a helix–loop–helix
transcription factor subfamily, which contains the potential
dimerization partners TFE3, TFEB, and TFEC. In mice, dominant negative,
but not recessive, mutations of
mitf
, produce
osteopetrosis, suggesting a functional requirement for other family
members. Mitf also has been found—and TFE3 has been suggested—to
modulate age-dependent changes in osteoclast function. This study
identifies cathepsin K as a transcriptional target of Mitf and TFE3 via
three consensus elements in the cathepsin K promoter. Additionally,
cathepsin K mRNA and protein were found to be deficient in
mitf
mutant osteoclasts, and overexpression of wild-type
Mitf dramatically up-regulated expression of endogenous
cathepsin K in cultured human osteoclasts. Cathepsin K promoter
activity was disrupted by dominant negative, but not recessive, mouse
alleles of
mitf
in a pattern that closely matches their
osteopetrotic phenotypes. This relationship between cathepsin K and the
Mitf family helps explain the phenotypic overlap of their corresponding
deficiencies in pycnodysostosis and osteopetrosis and identifies likely
regulators of cathepsin K expression in bone homeostasis and human
malignancy.
Identification of multiple quantitative trait loci linked to prion disease incubation period in mice
Authors: Lloyd Sarah E. , Onwuazor Obia N. , Beck Jonathan A. , Mallinson Gary , Farrall Martin , Targonski Paul , Collinge John , Fisher Elizabeth M. C.
Published: July 2002

Polymorphisms in the prion protein gene are known to affect prion disease incubation times and susceptibility in humans and mice. However, studies with inbred lines of mice show that large differences in incubation times occur even with the same amino acid sequence of the prion protein, suggesting that other genes may contribute to the observed variation. To identify these loci we analyzed 1,009 animals from an F2 intercross between two strains of mice, CAST/Ei and NZW/OlaHSd, with significantly different incubation periods when challenged with RML scrapie prions. Interval mapping identified three highly significantly linked regions on chromosomes 2, 11, and 12; composite interval mapping suggests that each of these regions includes multiple linked quantitative trait loci. Suggestive evidence for linkage was obtained on chromosomes 6 and 7. The sequence conservation between the mouse and human genome suggests that identification of mouse prion susceptibility alleles may have direct relevance to understanding human susceptibility to bovine spongiform encephalopathy (BSE) infection, as well as identifying key factors in the molecular pathways of prion pathogenesis. However, the demonstration of other major genetic effects on incubation period suggests the need for extreme caution in interpreting estimates of variant Creutzfeldt–Jakob disease epidemic size utilizing existing epidemiological models.
RNA virus error catastrophe: Direct molecular test by using ribavirin
Authors: Crotty Shane , Cameron Craig E. , Andino Raul
Published: July 2002

RNA viruses evolve rapidly. One source of this ability to rapidly
change is the apparently high mutation frequency in RNA virus
populations. A high mutation frequency is a central tenet of the
quasispecies theory. A corollary of the quasispecies theory postulates
that, given their high mutation frequency, animal RNA viruses may be
susceptible to error catastrophe, where they undergo a sharp drop in
viability after a modest increase in mutation frequency. We recently
showed that the important broad-spectrum antiviral drug ribavirin
(currently used to treat hepatitis C virus infections, among others) is
an RNA virus mutagen, and we proposed that ribavirin's antiviral
effect is by forcing RNA viruses into error catastrophe. However, a
direct demonstration of error catastrophe has not been made for
ribavirin or any RNA virus mutagen. Here we describe a direct
demonstration of error catastrophe by using ribavirin as the mutagen
and poliovirus as a model RNA virus. We demonstrate that ribavirin's
antiviral activity is exerted directly through lethal mutagenesis of
the viral genetic material. A 99.3% loss in viral genome infectivity
is observed after a single round of virus infection in ribavirin
concentrations sufficient to cause a 9.7-fold increase in mutagenesis.
Compiling data on both the mutation levels and the specific
infectivities of poliovirus genomes produced in the presence of
ribavirin, we have constructed a graph of error catastrophe showing
that normal poliovirus indeed exists at the edge of viability. These
data suggest that RNA virus mutagens may represent a promising new
class of antiviral drugs.
Life-long sustained mortality advantage of siblings of centenarians
Authors: Perls Thomas T. , Wilmoth John , Levenson Robin , Drinkwater Maureen , Cohen Melissa , Bogan Hazel , Joyce Erin , Brewster Stephanie , Kunkel Louis , Puca Annibale
Published: July 2002

Although survival to old age is known to have strong environmental and behavioral components, mortality differences between social groups tend to diminish or even disappear at older ages. Hypothesizing that surviving to extreme old age entails a substantial familial predisposition for longevity, we analyzed the pedigrees of 444 centenarian families in the United States. These pedigrees included 2,092 siblings of centenarians, whose survival was compared with 1900 birth cohort survival data from the U.S. Social Security Administration. Siblings of centenarians experienced a mortality advantage throughout their lives relative to the U.S. 1900 cohort. Female siblings had death rates at all ages about one-half the national level; male siblings had a similar advantage at most ages, although diminished somewhat during adolescence and young adulthood. Relative survival probabilities for these siblings increase markedly at older ages, reflecting the cumulative effect of their mortality advantage throughout life. Compared with the U.S. 1900 cohort, male siblings of centenarians were at least 17 times as likely to attain age 100 themselves, while female siblings were at least 8 times as likely.
MEK kinase 1 gene disruption alters cell migration and c-Jun NH
2
-terminal kinase regulation but does not cause a measurable defect in NF-κB activation
Authors: Yujiri Toshiaki , Ware Margaret , Widmann Christian , Oyer Ryan , Russell David , Chan Edward , Zaitsu Yuzuru , Clarke Penny , Tyler Kenneth , Oka Yoshitomo , Fanger Gary R. , Henson Peter , Johnson Gary L.
Published: July 2002

MEK kinase 1 (MEKK1) is a 196-kDa mitogen-activated protein kinase (MAPK) kinase kinase that, in addition to regulating the c-Jun NH
2
-terminal kinase (JNK) pathway, is involved in the control of cell motility. MEKK1
−/−
mice are defective in eyelid closure, a TGFα-directed process involving the migration of epithelial cells. MEKK1 expression in epithelial cells stimulates lamellipodia formation, a process required for cell movement. In addition, mouse embryo fibroblasts derived from MEKK1
−/−
mice are inhibited in their migration relative to MEKK1
+/+
fibroblasts. MEKK1 is required for JNK but not NF-κB activation in response to virus infection, microtubule disruption, and stimulation of embryonic stem cells with lysophosphatidic acid. MEKK1 is not required for TNFα or IL-1 regulation of JNK or NF-κB activation in macrophages or fibroblasts. Thus, MEKK1 senses microtubule integrity, contributes to the regulation of fibroblast and epithelial cell migration, and is required for activation of JNK but not NF-κB in response to selected stress stimuli.
Noninvasive gene targeting to the brain
Authors: Shi Ningya , Pardridge William M.
Published: July 2002

Gene therapy of the brain is hindered by the presence of the blood–brain barrier (BBB), which prevents the brain uptake of bloodborne gene formulations. Exogenous genes have been expressed in the brain after invasive routes of administration, such as craniotomy or intracarotid arterial infusion of noxious agents causing BBB disruption. The present studies describe the expression of an exogenous gene in brain after noninvasive i.v. administration of a 6- to 7-kb expression plasmid encoding either luciferase or β-galactosidase packaged in the interior of neutral pegylated immunoliposomes. The latter are conjugated with the OX26 mAb to the rat transferrin receptor, which enables targeting of the plasmid DNA to the brain via the endogenous BBB transferrin receptor. Unlike cationic liposomes, this neutral liposome formulation is stable in blood and does not result in selective entrapment in the lung. Luciferase gene expression in the brain peaks at 48 h after a single i.v. administration of 10 μg of plasmid DNA per adult rat, a dose that is 30- to 100-fold lower than that used for gene expression in rodents with cationic liposomes. β-Galactosidase histochemistry demonstrated gene expression throughout the central nervous system, including neurons, choroid plexus epithelium, and the brain microvasculature. In conclusion, widespread gene expression in the brain can be achieved by using a formulation that does not employ viruses or cationic liposomes, but instead uses endogenous receptor-mediated transport pathways at the BBB.
BACE2, a β-secretase homolog, cleaves at the β site and within the amyloid-β region of the amyloid-β precursor protein
Authors: Farzan Michael , Schnitzler Christine E. , Vasilieva Natalya , Leung Doris , Choe Hyeryun
Published: July 2002

Production of amyloid-β protein (Aβ) is initiated by a β-secretase that cleaves the Aβ precursor protein (APP) at the N terminus of Aβ (the β site). A recently identified aspartyl protease, BACE, cleaves the β site and at residue 11 within the Aβ region of APP. Here we show that BACE2, a BACE homolog, cleaves at the β site and more efficiently at a different site within Aβ. The Flemish missense mutation of APP, implicated in a form of familial Alzheimer's disease, is adjacent to this latter site and markedly increases Aβ production by BACE2 but not by BACE. BACE and BACE2 respond identically to conservative β-site mutations, and alteration of a common active site Arg inhibits β-site cleavage but not cleavage within Aβ by both enzymes. These data suggest that BACE2 contributes to Aβ production in individuals bearing the Flemish mutation, and that selective inhibition of these highly similar proteases may be feasible and therapeutically advantageous.
Binding of cyclin-dependent kinases to ORC and Cdc6p regulates the chromosome replication cycle
Authors: Weinreich Michael , Liang Chun , Chen Hsu-Hsin , Stillman Bruce
Published: July 2002

Cdc6p and the origin recognition complex (ORC) are essential
for assembly of a pre-replicative complex (preRC) at origins of
replication, before the initiation of DNA synthesis. In the absence of
Cdc6p, cells fail to initiate DNA replication and undergo a
“reductional” mitosis, in which the unreplicated chromosomes are
randomly segregated to the spindle poles. We show here that the cells
harboring a mutation in the essential Cdc6p Walker A-box arrest in late
mitosis, probably at anaphase. This cell cycle block requires either
the three Cdc28p phosphorylation sites within the N terminus of Cdc6p
or a short region (aa 8–17) that contains a Cy (Cyclin) interaction
sequence. These same two Cdc6p mutants that allow a reductional mitosis
are defective in binding Cdc28p kinase. In addition to Cdc6p, ORC also
binds to cyclin-dependent kinases (CDKs). Interestingly, Sic1p, a CDK
inhibitor protein, blocked the S phase-specific Cdc28p-Clb5p kinase
from interacting with ORC, but did not prevent the
G
1
-specific Cdc28p-Cln2p kinase–ORC interaction. We
suggest that ORC, Cdc6p, and Sic1p bind to different CDKs in a cell
cycle-dependent manner to temporally regulate events that
(
i
) allow preRC formation after mitosis,
(
ii
) prevent mitosis before DNA replication can occur,
and (
iii
) promote initiation of DNA replication.
Learning and memory
Authors: Okano Hideyuki , Hirano Tomoo , Balaban Evan
Published: July 2002

Memory is one of the most fundamental mental processes.
Neuroscientists study this process by using extremely diverse
strategies. Two different approaches aimed at understanding learning
and memory were introduced in this symposium. The first focuses on the
roles played by synaptic plasticity, especially in long-term depression
in the cerebellum in motor learning, and its regulatory mechanism. The
second approach uses an elegant chick-quail transplantation system on
defined brain regions to study how neural populations interact in
development to form behaviorally important neural circuits and to
elucidate neurobiological correlates of perceptual and motor
predispositions.
Sonic hedgehog restricts adhesion and migration of neural crest cells independently of the Patched- Smoothened-Gli signaling pathway
Authors: Testaz Sandrine , Jarov Artem , Williams Kevin P. , Ling Leona E. , Koteliansky Victor E. , Fournier-Thibault Claire , Duband Jean-Loup
Published: July 2002

In the vertebrate embryo, neural cell types are organized spatially along the dorsoventral axis of the neural tube and differ by expression of cell-intrinsic determinants and by their adhesive and locomotory properties. Thus, dorsally, neural crest cells (NCC) show a strong propensity to disperse and migrate, whereas cells situated ventrally are highly cohesive and poorly motile. Members of the bone morphogenetic proteins have been shown to exert a dual role in the specification of dorsal neuroepithelial cells and in the dispersion of NCCs. To test whether Sonic hedgehog (Shh), another signaling molecule involved in the patterning of the ventral neural tube, might also contribute to the control of the adhesive and migratory potential of neuroepithelial cells, we analyzed the effect of ectopic Shh on NCC dispersion from neural tube explants cultured
in vitro
. The addition of Shh to the migration substrate of NCC caused inhibition of their dispersion. The effect of Shh on cell migration was reversible and was not accounted for by alterations of the specification, delamination, proliferation, and survival of NCCs but could be essentially attributed to a decreased cell-substrate adhesion mediated by integrins. In addition, Shh activity on cell migration was mediated by a specific N-terminal region of the molecule and was independent from the signaling cascade elicited by the Patched-Smoothened receptor and involving the Gli transcription factors. Our study therefore reveals an unanticipated role for Shh in regulating adhesion and migration of neuroepithelial cells that is discernable from its inductive, mitogenic, and trophic functions.
Increased ability for selection of zidovudine resistance in a distinct class of wild-type HIV-1 from drug-naive persons
Authors: García-Lerma J. Gerardo , Nidtha Soumya , Blumoff Katherine , Weinstock Hillard , Heneine Walid
Published: July 2002

Transmission of HIV-1 with reduced susceptibility to antiretroviral drugs raises public health concerns. Through surveillance of drug-resistant HIV-1 in 603 treatment-naïve, recently diagnosed HIV-1-infected persons, we identified a distinct group of viruses that have mutations at codon 215 of the reverse transcriptase (RT) gene that are different from either the wild-type (WT) T or the zidovudine (AZT)-selected T215Y/F. These mutations included 215D/C/S and were found in 20 patients (3.3%). The 215D, 215C, and 215S mutations differ from 215Y by a 1-nt change compared with 2 nt for the WT T215 and likely represent revertants of 215Y. These viruses all were found to have WT susceptibility to AZT, and all replicated efficiently as WT HIV-1
T215
. However, differences in fitness among HIV-1
215D
, HIV-1
215C
, and HIV-1
215S
were seen when RT backgrounds were changed, demonstrating a role of the RT background in the selection of these revertants.
In vitro
selection with AZT showed that HIV-1
215D
and HIV-1
215C
acquired 215Y more rapidly than did WT HIV-1
T215
, likely reflecting the need for only 1-nt change to evolve to 215Y. Our study demonstrates that HIV-1 with unusual mutations at codon 215 replicate efficiently, have WT susceptibility, and are commonly found in treatment-naïve persons. The increased ability for selecting resistance mutations defines this class of WT HIV-1 and highlights the higher potential of these viruses to compromise the efficacy of antiretroviral therapy.
A distinct glycoform of CD44 is an L-selectin ligand on human hematopoietic cells
Authors: Dimitroff Charles J. , Lee Jack Y. , Fuhlbrigge Robert C. , Sackstein Robert
Published: July 2002

We previously have obtained operational evidence of a hematopoietic cell L-selectin ligand expressed on normal human hematopoietic cells and on leukemic blasts. Using a technique developed in our laboratory for analyzing and identifying adhesion molecules, we show here that hematopoietic cell L-selectin ligand is a specialized glycoform of CD44. This L-selectin ligand activity of CD44 requires sialofucosylated N-linked glycans and is sulfation-independent. These data provide important insights on the structural biology of CD44 and reveal a role for this protein as an L-selectin ligand on human hematopoietic cells.
A domain unique to plant RanGAP is responsible for its targeting to the plant nuclear rim
Authors: Rose Annkatrin , Meier Iris
Published: July 2002

Ran is a small signaling GTPase that is involved in
nucleocytoplasmic transport. Two additional functions of animal Ran in
the formation of spindle asters and the reassembly of the nuclear
envelope in mitotic cells have been recently reported. In contrast to
Ras or Rho, Ran is not associated with membranes. Instead, the spatial
sequestering of its accessory proteins, the Ran GTPase-activating
protein RanGAP and the nucleotide exchange factor RCC1, appears to
define the local concentration of RanGTP vs. RanGDP involved in
signaling. Mammalian RanGAP is bound to the nuclear pore by a mechanism
involving the attachment of small ubiquitin-related modifier
protein (SUMO) to its C terminus and the subsequent binding of
the SUMOylated domain to the nucleoporin Nup358. Here we show that
plant RanGAP utilizes a different mechanism for nuclear envelope
association, involving a novel targeting domain that appears to be
unique to plants. The N-terminal WPP domain is highly conserved among
plant RanGAPs and the small, plant-specific nuclear envelope-associated
protein MAF1, but not present in yeast or animal RanGAP. Confocal laser
scanning microscopy of green fluorescent protein (GFP) fusion proteins
showed that it is necessary for RanGAP targeting and sufficient to
target the heterologous protein GFP to the plant nuclear rim. The
highly conserved tryptophan and proline residues of the WPP motif are
necessary for its function. The 110-aa WPP domain is the first
nuclear-envelope targeting domain identified in plants. Its fundamental
difference to its mammalian counterpart implies that different
mechanisms have evolved in plants and animals to anchor RanGAP at the
nuclear surface.
Phosphatidylinositol 4,5-bisphosphate stimulates vesicle formation from liposomes by brain cytosol
Authors: Kinuta Masahiro , Yamada Hiroshi , Abe Tadashi , Watanabe Masami , Li Shun-Ai , Kamitani Akihiro , Yasuda Tatsuji , Matsukawa Takashi , Kumon Hiromi , Takei Kohji
Published: July 2002

As a step toward the elucidation of mechanisms in vesicle budding, a cell-free assay that measures cytosol-induced vesicle generation from liposomes was established. This assay then was used to explore the role of phosphoinositides in vesicle formation. Liposomes incubated with brain cytosol in the presence of ATP and GTP massively generated small vesicles, as assessed both quantitatively and qualitatively by a dynamic light-scattering assay. Both ATP and GTP were required. Vesicle formation was inhibited greatly by the immunodepletion of dynamin 1 from the cytosol, indicating a major contribution of this GTPase in this reaction and suggesting that it mimics endocytic vesicle fission. Increasing the concentration of
l
-α-phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P
2
] but not of
l
-α-phosphatidylinositol 4-monophosphate or
l
-α-phosphatidylinositol in the lipid membranes enhanced vesicle formation. Lipid analysis revealed rapid degradation of PtdIns(4,5)P
2
to
l
-α-phosphatidylinositol during the incubation with the reaction reaching a maximum within 5 sec, whereas vesicle formation proceeded with a longer time course. PtdIns(4,5)P
2
degradation was independent of vesicle formation and occurred also in the presence of guanosine 5′-
O
-(thiotriphosphate), where few vesicle formations occurred. These results suggest that PtdIns(4,5)P
2
plays a critical role in the early step of vesicle formation, possibly in the recruitment of coats and fission factors to membranes.
High-level production of recombinant human lysosomal acid alpha-glucosidase in Chinese hamster ovary cells which targets to heart muscle and corrects glycogen accumulation in fibroblasts from patients with Pompe disease.
Authors: Van Hove J L , Yang H W , Wu J Y , Brady R O , Chen Y T
Published: July 2002

Infantile Pompe disease is a fatal genetic muscle disorder caused by a deficiency of acid alpha-glucosidase, a glycogen-degrading lysosomal enzyme. We constructed a plasmid containing a 5'-shortened human acid alpha-glucosidase cDNA driven by the cytomegalovirus promoter, as well as the aminoglycoside phosphotransferase and dihydrofolate reductase genes. Following transfection in dihydrofolate reductase-deficient Chinese hamster ovary cells, selection with Geneticin, and amplification with methotrexate, a cell line producing high levels of the alpha-glucosidase was established. In 48 hr, the cells cultured in Iscove's medium with 5 mM butyrate secreted 110-kDa precursor enzyme that accumulated to 91 micrograms.ml-1 in the medium (activity, > 22.6 mumol.hr-1.ml-1). This enzyme has a pH optimum similar to that of the mature form, but a lower Vmax and Km for 4-methylumbelliferyl-alpha-D-glucoside. It is efficiently taken up by fibroblasts from Pompe patients, restoring normal levels of acid alpha-glucosidase and glycogen. The uptake is blocked by mannose 6-phosphate. Following intravenous injection, high enzyme levels are seen in heart and liver. An efficient production system now exists for recombinant human acid alpha-glucosidase targeted to heart and capable of correcting fibroblasts from patients with Pompe disease.
The recombinant proregion of transforming growth factor beta1 (latency-associated peptide) inhibits active transforming growth factor beta1 in transgenic mice.
Authors: Böttinger E P , Factor V M , Tsang M L , Weatherbee J A , Kopp J B , Qian S W , Wakefield L M , Roberts A B , Thorgeirsson S S , Sporn M B
Published: July 2002

All three isoforms of transforming growth factors beta (TGF-betal, TGF-beta2, and TGF-beta3) are secreted as latent complexes and activated extracellularly, leading to the release of the mature cytokines from their noncovalently associated proregions, also known as latency-associated peptides (LAPs). The LAP region of TGF-beta1 was expressed in a baculovirus expression system and purified to homogeneity. In vitro assays of growth inhibition and gene induction mediated by TGF-beta3 demonstrate that recombinant TGF-beta1 LAP is a potent inhibitor of the activities of TGF-betal, -beta2, and -beta3. Effective dosages of LAP for 50% neutralization of TGF-beta activities range from 4.7- to 80-fold molar excess depending on the TGF-beta isoform and activity examined. Using 125I-labeled LAP, we show that the intraperitoneal application route is effective for systemic administration of LAP. Comparison of concentrations of LAP in tissues shows a homogenous pattern in most organs with the exception of heart and muscle, in which levels of LAP are 4- to 8-fold lower. In transgenic mice with elevated hepatic levels of bioactive TGF-betal, treatment with recombinant LAP completely reverses suppression of the early proliferative response induced by TGF-beta1 in remnant livers after partial hepatectomy. The results suggest that recombinant LAP is a potent inhibitor of bioactive TGF-beta both in vitro and in vivo, after intraperitoneal administration. Recombinant LAP should be a useful tool for novel approaches to study and therapeutically modulate pathophysiological processes mediated by TGF-beta3.
Processing of the leader mRNA plays a major role in the induction of thrS expression following threonine starvation in Bacillus subtilis.
Authors: Condon C , Putzer H , Grunberg-Manago M
Published: July 2002

The threonyl-tRNA synthetase gene, thrS, is a member of a family of Gram-positive genes that are induced following starvation for the corresponding amino acid by a transcriptional antitermination mechanism involving the cognate uncharged tRNA. Here we show that an additional level of complexity exists in the control of the thrS gene with the mapping of an mRNA processing site just upstream of the transcription terminator in the thrS leader region. The processed RNA is significantly more stable than the full-length transcript. Under nonstarvation conditions, or following starvation for an amino acid other than threonine, the full-length thrS mRNA is more abundant than the processed transcript. However, following starvation for threonine, the thrS mRNA exists primarily in its cleaved form. This can partly be attributed to an increased processing efficiency following threonine starvation, and partly to a further, nonspecific increase in the stability of the processed transcript under starvation conditions. The increased stability of the processed RNA contributes significantly to the levels of functional RNA observed under threonine starvation conditions, previously attributed solely to antitermination. Finally, we show that processing is likely to occur upstream of the terminator in the leader regions of at least four other genes of this family, suggesting a widespread conservation of this phenomenon in their control.
Intermolecular disulfide bonds stabilize VirB7 homodimers and VirB7/VirB9 heterodimers during biogenesis of the Agrobacterium tumefaciens T-complex transport apparatus.
Authors: Spudich G M , Fernandez D , Zhou X R , Christie P J
Published: July 2002

The Agrobacterium tumefaciens VirB7 lipoprotein contributes to the stabilization of VirB proteins during biogenesis of the putative T-complex transport apparatus. Here, we report that stabilization of VirB7 itself is correlated with its ability to form disulfide cross-linked homodimers via a reactive Cys-24 residue. Three types of beta-mercaptoethanol-dissociable complexes were visualized with VirB7 and/or a VirB7::PhoA41 fusion protein: (i) a 9-kDa complex corresponding in size to a VirB7 homodimer, (ii) a 54-kDa complex corresponding in size to a VirB7/VirB7::PhoA41 mixed dimer, and (iii) a 102-kDa complex corresponding to a VirB7::PhoA41 homodimer. A VirB7C24S mutant protein was immunologically undetectable, whereas the corresponding VirB7C24S::PhoA41 derivative accumulated to detectable levels but failed to form dissociable homodimers or mixed dimers with wild-type VirB7. We further report that VirB7-dependent stabilization of VirB9 is correlated with the ability of these two proteins to dimerize via formation of a disulfide bridge between reactive Cys-24 and Cys-262 residues, respectively. Two types of dissociable complexes were visualized: (i) a 36-kDa complex corresponding in size to a VirB7/VirB9 heterodimer and (ii) an 84-kDa complex corresponding in size to a VirB7/VirB9::PhoA293 heterodimer. A VirB9C262S mutant protein was immunologically undetectable, whereas the corresponding VirB9C262S::PhoA293 derivative accumulated to detectable levels but failed to form dissociable heterodimers with wild-type VirB7. Taken together, these results support a model in which the formation of disulfide cross-linked VirB7 dimers represent critical early steps in the biogenesis of the T-complex transport apparatus.
Creation of a novel protein-coding region at the RNA level in black pine chloroplasts: the pattern of RNA editing in the gymnosperm chloroplast is different from that in angiosperms.
Authors: Wakasugi T , Hirose T , Horihata M , Tsudzuki T , Kössel H , Sugiura M
Published: July 2002

The phenomenon of RNA editing has been found to occur in chloroplasts of several angiosperm plants. Comparative analysis of the entire nucleotide sequence of a gymnosperm [Pinus thunbergii (black pine)] chloroplast genome allowed us to predict several potential editing sites in its transcripts. Forty-nine such sites from 14 genes/ORFs were analyzed by sequencing both cDNAs from the transcripts and the corresponding chloroplast DNA regions, and 26 RNA editing sites were identified in the transcripts from 12 genes/ORFs, indicating that chloroplast RNA editing is not restricted to angiosperms but occurs in the gymnosperm, too. All the RNA editing events are C-to-U conversions; however, many new codon substitutions and creation of stop codons that have not so far been reported in angiosperm chloroplasts were observed. The most striking is that two editing events result in the creation of an initiation and a stop codon within a single transcript, leading to the formation of a new reading frame of 33 codons. The predicted product is highly homologous to that deduced from the ycf7 gene (ORF31), which is conserved in the chloroplast genomes of many other plant species.
Mice with disrupted GM2/GD2 synthase gene lack complex gangliosides but exhibit only subtle defects in their nervous system.
Authors: Takamiya K , Yamamoto A , Furukawa K , Yamashiro S , Shin M , Okada M , Fukumoto S , Haraguchi M , Takeda N , Fujimura K , Sakae M , Kishikawa M , Shiku H , Furukawa K , Aizawa S
Published: July 2002

Gangliosides, sialic acid-containing glycosphingolipids, are abundant in the vertebrate (mammalian) nervous system. Their composition is spatially and developmentally regulated, and gangliosides have been widely believed to lay essential roles in establishment of the nervous system, especially in neuritogenesis and synaptogenesis. However, this has never been tested directly. Here we report the generation of mice with a disrupted beta 1,4-N-acetylgalactosaminyltransferase (GM2/GD2 synthase; EC 2.4.1.92) gene. The mice lacked all complex gangliosides. Nevertheless, they did not show any major histological defects in their nervous systems or in gross behavior. Just a slight reduction in the neural conduction velocity from the tibial nerve to the somatosensory cortex, but not to the lumbar spine, was detected. These findings suggest that complex gangliosides are required in neuronal functions but not in the morphogenesis and organogenesis of the brain. The higher levels of GM3 and GD3 expressed in the brains of these mutant mice may be able to compensate for the lack of complex gangliosides.
Integrin regulation of c-Abl tyrosine kinase activity and cytoplasmic–nuclear transport
Authors: Lewis Jean M. , Baskaran Rajasekaran , Taagepera Salme , Schwartz Martin A. , Wang Jean Y. J.
Published: July 2002

The product of the c-
abl
protooncogene is a
nonreceptor tyrosine kinase found in both the cytoplasm and the
nucleus. We report herein that cell adhesion regulates the kinase
activity and subcellular localization of c-Abl. When fibroblastic cells
are detached from the extracellular matrix, kinase activity of both
cytoplasmic and nuclear c-Abl decreases, but there is no detectable
alteration in the subcellular distribution. Upon adhesion to the
extracellular matrix protein fibronectin, a transient recruitment of a
subset of c-Abl to early focal contacts is observed coincident with the
export of c-Abl from the nucleus to the cytoplasm. The cytoplasmic pool
of c-Abl is reactivated within 5 min of adhesion, but the nuclear c-Abl
is reactivated after 30 min, correlating closely with its return to the
nucleus and suggesting that the active nuclear c-Abl originates in the
cytoplasm. In quiescent cells where nuclear c-Abl activity is low, the
cytoplasmic c-Abl is similarly regulated by adhesion but the nuclear
c-Abl is not activated upon cell attachment. These results show that
c-Abl activation requires cell adhesion and that this tyrosine kinase
can transmit integrin signals to the nucleus where it may function to
integrate adhesion and cell cycle signals.
DNA binding specificity of two homeodomain proteins in vitro and in Drosophila embryos.
Authors: Walter J , Biggin M D
Published: July 2002

In previous experiments, the homeodomain proteins even-skipped and fushi-tarazu were found to UV cross-link to a surprisingly wide array of DNA sites in living Drosophila embryos. We now show that UV cross-linking gives a highly accurate measure of DNA binding by these proteins. In addition, the binding of even-skipped and fushi-tarazu proteins has been measured in vitro to the same DNA fragments that were examined in vivo. This analysis shows that these proteins have broad DNA recognition properties in vitro that are likely to be important determinants of their distribution on DNA in vivo, but it also shows that in vitro DNA binding specificity alone is not sufficient to explain the distribution of these proteins in embryos.
Multiple-complete-digest restriction fragment mapping: Generating sequence-ready maps for large-scale DNA sequencing
Authors: Wong Gane K.-S. , Yu Jun , Thayer Edward C. , Olson Maynard V.
Published: July 2002

Multiple-complete-digest mapping is a DNA mapping technique based on complete-restriction-digest fingerprints of a set of clones that provides highly redundant coverage of the mapping target. The maps assembled from these fingerprints order both the clones and the restriction fragments. Maps are coordinated across three enzymes in the examples presented. Starting with yeast artificial chromosome contigs from the 7q31.3 and 7p14 regions of the human genome, we have produced cosmid-based maps spanning more than one million base pairs. Each yeast artificial chromosome is first subcloned into cosmids at a redundancy of ×15–30. Complete-digest fragments are electrophoresed on agarose gels, poststained, and imaged on a fluorescent scanner. Aberrant clones that are not representative of the underlying genome are rejected in the map construction process. Almost every restriction fragment is ordered, allowing selection of minimal tiling paths with clone-to-clone overlaps of only a few thousand base pairs. These maps demonstrate the practicality of applying the experimental and software-based steps in multiple-complete-digest mapping to a target of significant size and complexity. We present evidence that the maps are sufficiently accurate to validate both the clones selected for sequencing and the sequence assemblies obtained once these clones have been sequenced by a “shotgun” method.
Rhizobium
gone native: Unexpected plasmid stability of indigenous
Rhizobium leguminosarum
Authors: Wernegreen Jennifer J. , Harding Ethelynda E. , Riley Margaret A.
Published: July 2002

Lateral transfer of bacterial plasmids is thought to play an important role in microbial evolution and population dynamics. However, this assumption is based primarily on investigations of medically or agriculturally important bacterial species. To explore the role of lateral transfer in the evolution of bacterial systems not under intensive, human-mediated selection, we examined the association of genotypes at plasmid-encoded and chromosomal loci of native
Rhizobium
, the nitrogen-fixing symbiont of legumes. To this end,
Rhizobium leguminosarum
strains nodulating sympatric species of native
Trifolium
were characterized genetically at plasmid-encoded symbiotic (
sym
) regions (nodulation AB and nodulation CIJT loci) and a repeated chromosomal locus not involved in the symbiosis with legumes. Restriction fragment length polymorphism analysis was used to distinguish genetic groups at plasmid and chromosomal loci. The correlation between major
sym
and chromosomal genotypes and the distribution of genotypes across host plant species and sampling location were determined using χ
2
analysis. In contrast to findings of previous studies, a strict association existed between major
sym
plasmid and chromosomal genetic groups, suggesting a lack of successful
sym
plasmid transfer between major
Rhizobium
chromosomal types. These data indicate that previous observations of
sym
plasmid transfer in agricultural settings may seriously overestimate the rates of successful conjugation in systems not impacted by human activities. In addition, a nonrandom distribution of
Rhizobium
genotypes across host plant species and sampling site demonstrates the importance of both factors in shaping
Rhizobium
population dynamics.
Detecting and characterizing
N
-acyl-homoserine lactone signal molecules by thin-layer chromatography
Authors: Shaw Paul D. , Ping Gao , Daly Sean L. , Cha Chung , Cronan John E. , Rinehart Kenneth L. , Farrand Stephen K.
Published: July 2002

Many Gram-negative bacteria regulate gene expression in response to their population size by sensing the level of acyl-homoserine lactone signal molecules which they produce and liberate to the environment. We have developed an assay for these signals that couples separation by thin-layer chromatography with detection using
Agrobacterium tumefaciens
harboring
lacZ
fused to a gene that is regulated by autoinduction. With the exception of
N
-butanoyl-
l
-homoserine lactone, the reporter detected acyl-homoserine lactones with 3-oxo-, 3-hydroxy-, and 3-unsubstituted side chains of all lengths tested. The intensity of the response was proportional to the amount of the signal molecule chromatographed. Each of the 3-oxo- and the 3-unsubstituted derivatives migrated with a unique mobility. Using the assay, we showed that some bacteria produce as many as five detectable signal molecules. Structures could be assigned tentatively on the basis of mobility and spot shape. The dominant species produced by
Pseudomonas syringae
pv.
tabaci
chromatographed with the properties of
N
-(3-oxohexanoyl)-
l
-homoserine lactone, a structure that was confirmed by mass spectrometry. An isolate of
Pseudomonas fluorescens
produced five detectable species, three of which had novel chromatographic properties. These were identified as the 3-hydroxy- forms of
N
-hexanoyl-,
N
-octanoyl-, and
N
-decanoyl-
l
-homoserine lactone. The assay can be used to screen cultures of bacteria for acyl-homoserine lactones, for quantifying the amounts of these molecules produced, and as an analytical and preparative aid in determining the structures of these signal molecules.
Wip1, a novel human protein phosphatase that is induced in response to ionizing radiation in a p53-dependent manner
Authors: Fiscella Michele , Zhang HongLiang , Fan Saijun , Sakaguchi Kazuyasu , Shen Songfa , Mercer W. Edward , Vande Woude George F. , O’Connor Patrick M. , Appella Ettore
Published: July 2002

Exposure of mammalian cells to ionizing radiation (IR) induces a complex array of cellular responses including cell cycle arrest and/or apoptosis. IR-induced G
1
arrest has been shown to depend on the presence of the tumor suppressor p53, which acts as a transcriptional activator of several genes. p53 also plays a role in the induction of apoptosis in response to DNA damage, and this pathway can be activated by both transcription-dependent and -independent mechanisms. Here we report the identification of a novel transcript whose expression is induced in response to IR in a p53-dependent manner, and that shows homology to the type 2C protein phosphatases. We have named this novel gene,
wip1. In vitro
, recombinant Wip1 displayed characteristics of a type 2C phosphatase, including Mg
2+
dependence and relative insensitivity to okadaic acid. Studies performed in several cell lines revealed that
wip1
accumulation following IR correlates with the presence of wild-type p53. The accumulation of
wip1
mRNA following IR was rapid and transient, and the protein was localized to the nucleus. Similar to
waf1,
ectopic expression of
wip1
in human cells suppressed colony formation. These results suggest that Wip1 might contribute to growth inhibitory pathways activated in response to DNA damage in a p53-dependent manner.
High level expression of p27
kip1
and cyclin D1 in some human breast cancer cells: Inverse correlation between the expression of p27
kip1
and degree of malignancy in human breast and colorectal cancers
Authors: Fredersdorf Steffen , Burns Jorges , Milne Alistair M. , Packham Graham , Fallis Lynn , Gillett Cheryl E. , Royds Janice A. , Peston David , Hall Peter A. , Hanby Andrew M. , Barnes Diana M. , Shousha Sami , O’Hare Mike J. , Lu Xin
Published: July 2002

The expression of cyclin-dependent kinase inhibitor p27
kip1
in human tumors and normal tissues was investigated using a panel of novel anti-p27
kip1
mAbs. An inverse correlation between expression of p27
kip1
and cell proliferation was generally observed after analyzing its expression in 25 different normal human tissues. In some highly proliferative human breast cancer cells, however, high level p27
kip1
expression was seen, indicating the existence of a mechanism by which some growing tumor cells may tolerate this inhibitor of cell cycle progression. Detailed studies demonstrated a correlation between the high level expression of p27
kip1
and cyclin D1 in human breast cancer cells. There was also an inverse correlation between the expression of p27
kip1
and the degree of tumor malignancy in human breast and colorectal cancers, indicating that p27
kip1
may be a useful prognostic marker in these cancers.
Differential behavior of curved DNA upon untwisting
Authors: Brukner Ivan , Belmaaza Abdellah , Chartrand Pierre
Published: July 2002

We have synthesized DNA segments with different handedness, twisting and radii of curvature, and have analyzed the effect of untwisting on them. The results indicate that the dynamic behavior of curved DNA upon untwisting is strongly determined by the initial sequence-dependent DNA trajectory. In particular, DNA with the same radii but with opposite handedness of superhelix twisting can show very different conformational responses to ethidium bromide untwisting. Upon treatment with ethidium bromide, right-handed superhelixes decrease their twist and increase the planarity of the superhelix, while left-handed superhelixes increase twisting and decrease their degree of planarity.
Bok is a pro-apoptotic Bcl-2 protein with restricted expression in reproductive tissues and heterodimerizes with selective anti-apoptotic Bcl-2 family members
Authors: Hsu Sheau Yu , Kaipia Antti , McGee E. , Lomeli Michelle , Hsueh Aaron J. W.
Published: July 2002

In the intracellular death program, hetero- and homodimerization of different anti- and pro-apoptotic Bcl-2-related proteins are critical in the determination of cell fate. From a rat ovarian fusion cDNA library, we isolated a new pro-apoptotic Bcl-2 gene, Bcl-2-related ovarian killer (Bok). Bok had conserved Bcl-2 homology (BH) domains 1, 2, and 3 and a C-terminal transmembrane region present in other Bcl-2 proteins, but lacked the BH4 domain found only in anti-apoptotic Bcl-2 proteins. In the yeast two-hybrid system, Bok interacted strongly with some (Mcl-1, BHRF1, and Bfl-1) but not other (Bcl-2, Bcl-xL, and Bcl-w) anti-apoptotic members. This finding is in direct contrast to the ability of other pro-apoptotic members (Bax, Bak, and Bik) to interact with all of the anti-apoptotic proteins. In addition, negligible interaction was found between Bok and different pro-apoptotic members. In mammalian cells, overexpression of Bok induced apoptosis that was blocked by the baculoviral-derived cysteine protease inhibitor P35. Cell killing induced by Bok was also suppressed following coexpression with Mcl-1 and BHRF1 but not with Bcl-2, further indicating that Bok heterodimerized only with selective anti-apoptotic Bcl-2 proteins. Northern blot analysis indicated that Bok was highly expressed in the ovary, testis and uterus.In situhybridization analysis localized Bok mRNA in granulosa cells, the cell type that underwent apoptosis during follicle atresia. Identification of Bok as a new pro-apoptotic Bcl-2 protein with restricted tissue distribution and heterodimerization properties could facilitate elucidation of apoptosis mechanisms in reproductive tissues undergoing hormone-regulated cyclic cell turnover.