bluuebunny/crossref_metadata_2025 huggingface dataset:
AID-GFP chimeric protein increases hypermutation of Ig genes with no evidence of nuclear localization
Authors: Rada Cristina , Jarvis John M. , Milstein César
Published: July 2002

Somatic hypermutation generates variants of antibody genes and underpins the affinity maturation of antibodies. It is restricted to the V-gene segments, and although it decays exponentially toward the 3′end, it includes recognizable hot spots. Although the detailed mechanism of hypermutation remains elusive, the process may take place in two separate stages, preferentially targeting G/Cs in the first and A/Ts in the second stage. It seems that MSH2 is involved in the second stage, and that activation induced deaminase (AID) is implicated in the control of hypermutation. The constitutively hypermutating cell line Ramos expresses AID, and we have prepared transfectants that express a chimeric AID-green fluorescent protein. The fluorescence is strongly detected in the cytoplasm but not in the nucleus. Yet, the chimeric protein increases the hypermutation rate either directly or, more likely, indirectly, by favoring the transport of AID into the nucleus. Thus, in Ramos, AID seems to be rate limiting. Unexpectedly, the proportion of deletions also is increased. The increase in mutation rate detected by a fast cytofluorimetric method based on the accumulation of sIgM-loss mutants correlates with the increase measured by mutations defined by sequence analysis. The higher mutation rate is largely explained by the higher proportion of mutated clones, indicating that AID controls the number of cells that undergo hypermutation but not the number of mutations that are incorporated in each mutation round.
Twentieth century sea level: An enigma
Authors: Munk Walter
Published: July 2002

Changes in sea level (relative to the moving crust) are associated with changes in ocean volume (mostly thermal expansion) and in ocean mass (melting and continental storage): ζ(
t
) = ζ
steric
(
t
) + ζ
eustatic
(
t
). Recent compilations of global ocean temperatures by Levitus and coworkers are in accord with coupled ocean/atmosphere modeling of greenhouse warming; they yield an increase in 20th century ocean heat content by 2 × 10
23
J (compared to 0.1 × 10
23
J of atmospheric storage), which corresponds to ζ
greenhouse
(2000) = 3 cm. The greenhouse-related rate is accelerating, with a present value ζ̇
greenhouse
(2000) ≈ 6 cm/century. Tide records going back to the 19th century show no measurable acceleration throughout the late 19th and first half of the 20th century; we take ζ̇
historic
= 18 cm/century. The Intergovernmental Panel on Climate Change attributes about 6 cm/century to melting and other eustatic processes, leaving a residual of 12 cm of 20th century rise to be accounted for. The Levitus compilation has virtually foreclosed the attribution of the residual rise to ocean warming (notwithstanding our ignorance of the abyssal and Southern Oceans): the historic rise started too early, has too linear a trend, and is too large. Melting of polar ice sheets at the upper limit of the Intergovernmental Panel on Climate Change estimates could close the gap, but severe limits are imposed by the observed perturbations in Earth rotation. Among possible resolutions of the enigma are: a substantial reduction from traditional estimates (including ours) of 1.5–2 mm/y global sea level rise; a substantial increase in the estimates of 20th century ocean heat storage; and a substantial change in the interpretation of the astronomic record.
Expression profiling reveals distinct sets of genes altered during induction and regression of cardiac hypertrophy
Authors: Friddle Carl J. , Koga Teiichiro , Rubin Edward M. , Bristow James
Published: July 2002

Although cardiac hypertrophy has been the subject of intensive investigation, regression of hypertrophy has been significantly less studied, precluding large-scale analysis of the relationship between these processes. In the present study, using pharmacological models of cardiac hypertrophy in mice, expression profiling was performed with fragments of more than 4,000 genes to characterize and contrast expression changes during induction and regression of hypertrophy. Administration of angiotensin II and isoproterenol by osmotic minipump produced increases in heart weight (15 and 45%, respectively) that returned to preinduction size after drug withdrawal. From multiple expression analyses of left ventricular RNA isolated at daily time-points during cardiac hypertrophy and regression, we identified sets of genes whose expression was altered at specific stages of this process. While confirming the participation of 25 genes or pathways previously shown to be altered by hypertrophy, a larger set of 30 genes was identified whose expression had not previously been associated with cardiac hypertrophy or regression. Of the 55 genes that showed reproducible changes during the time course of induction and regression, 32 genes were altered only during induction, and 8 were altered only during regression. This study identified both known and novel genes whose expression is affected at different stages of cardiac hypertrophy and regression and demonstrates that cardiac remodeling during regression utilizes a set of genes that are distinct from those used during induction of hypertrophy.
Identification of four candidate cGMP targets in
Dictyostelium
Authors: Goldberg Jonathan M. , Bosgraaf Leonard , Van Haastert Peter J. M. , Smith Janet L.
Published: July 2002

In
Dictyostelium
, a transient increase in intracellular cGMP is important for cytoskeletal rearrangements during chemotaxis. There must be cGMP-binding proteins in
Dictyostelium
that regulate key cytoskeletal components after treatment with chemoattractants, but to date, no such proteins have been identified. Using a bioinformatics approach, we have found four candidate cGMP-binding proteins (GbpA–D). GbpA and -B have two tandem cGMP-binding sites downstream of a metallo β-lactamase domain, a superfamily that includes cAMP phosphodiesterases. GbpC contains the following nine domains (in order): leucine-rich repeats, Ras, MEK kinase, Ras guanine nucleotide exchange factor N-terminal (RasGEF-N), DEP, RasGEF, cGMP-binding, GRAM, and a second cGMP-binding domain. GbpD is related to GbpC, but is much shorter; it begins with the RasGEF-N domain, and lacks the DEP domain. Disruption of the
gbp
C gene results in loss of all high-affinity cGMP-binding activity present in the soluble cellular fraction.
Gbp
C mRNA levels increase dramatically 8 h after starvation is initiated.
Gbp
A, -B, and -D mRNA levels show less dramatic changes, with
gbp
A mRNA levels highest 4 h into starvation,
gbp
B mRNA levels highest in vegetative cells, and
gbp
D levels highest at 8 h. The identification of these genes is the first step in a molecular approach to studying downstream effects of cGMP signaling in
Dictyostelium
.
Ribonucleoprotein infrastructure regulating the flow of genetic information between the genome and the proteome
Authors: Keene Jack D.
Published: July 2002

Following transcription and splicing, each mRNA of a mammalian cell passes into the cytoplasm where its fate is in the hands of a complex network of ribonucleoproteins (mRNPs). The success or failure of a gene to be expressed depends on the performance of this mRNP infrastructure. The entry, gating, processing, and transit of each mRNA through an mRNP network helps determine the composition of a cell's proteome. The machinery that regulates storage, turnover, and translational activation of mRNAs is not well understood, in part, because of the heterogeneous nature of mRNPs. Recently, subsets of cellular mRNAs clustered as members of mRNP complexes have been identified by using antibodies reactive with RNA-binding proteins, including ELAV/Hu, eIF-4E, and poly(A)-binding proteins. Cytoplasmic ELAV/Hu proteins are involved in the stability and translation of early response gene (ERG) transcripts and are expressed predominately in neurons. mRNAs recovered from ELAV/Hu mRNP complexes were found to have similar sequence elements, suggesting a common structural linkage among them. This approach opens the possibility of identifying transcripts physically clustered
in vivo
that may have similar fates or functions. Moreover, the proteins encoded by physically organized mRNAs may participate in the same biological process or structural outcome, not unlike operons and their polycistronic mRNAs do in prokaryotic organisms. Our goal is to understand the organization and flow of genetic information on an integrative systems level by analyzing the collective properties of proteins and mRNAs associated with mRNPs
in vivo
.
Remodeling of the postnatal mouse testis is accompanied by dramatic changes in stem cell number and niche accessibility
Authors: Shinohara Takashi , Orwig Kyle E. , Avarbock Mary R. , Brinster Ralph L.
Published: July 2002

Little is known about stem cell biology or the specialized environments or niches believed to control stem cell renewal and differentiation in self-renewing tissues of the body. Functional assays for stem cells are available only for hematopoiesis and spermatogenesis, and the microenvironment, or niche, for hematopoiesis is relatively inaccessible, making it difficult to analyze donor stem cell colonization events in recipients. In contrast, the recently developed spermatogonial stem cell assay system allows quantitation of individual colonization events, facilitating studies of stem cells and their associated microenvironment. By using this assay system, we found a 39-fold increase in male germ-line stem cells during development from birth to adult in the mouse. However, colony size or area of spermatogenesis generated by neonate and adult stem cells, 2–3 months after transplantation into adult tubules, was similar (∼0.5 mm2). In contrast, the microenvironment in the immature pup testis was 9.4 times better than adult testis in allowing colonization events, and the area colonized per donor stem cell, whether from adult or pup, was about 4.0 times larger in recipient pups than adults. These factors facilitated the restoration of fertility by donor stem cells transplanted to infertile pups. Thus, our results demonstrate that stem cells and their niches undergo dramatic changes in the postnatal testis, and the microenvironment of the pup testis provides a more hospitable environment for transplantation of male germ-line stem cells.
Ordered recruitment of histone acetyltransferases and the TRAP/Mediator complex to thyroid hormone-responsive promoters
in vivo
Authors: Sharma Dipali , Fondell Joseph D.
Published: July 2002

Transcriptional coactivators implicated in gene activation by the thyroid hormone receptor (TR) include members of the p160/steroid receptor coactivator (SRC) family of proteins, p300, and the multisubunit TR-associated protein (TRAP)/Mediator complex. We investigated the temporal recruitment of these cofactors to mammalian thyroid hormone (T3)-responsive promoters
in vivo
. We show that upon T3 treatment, TR recruits all three types of coactivators to specific promoters in at least two sequential steps: p160/SRC proteins and p300 are recruited first and rapidly induce histone acetylation, followed by the recruitment of the TRAP/Mediator complex. Interestingly, inhibition of histone deacetylase activity with trichostatin A elicited a more rapid promoter recruitment of the TRAP/Mediator complex but not p160/SRC proteins. T3-dependent gene expression assays indicate that all three coactivators are targeted to a promoter before significant activation occurs. These findings thus suggest that histone acetylation may be a prerequisite for TRAP/Mediator recruitment and function at specific T3-responsive mammalian promoters.
MEKK1 plays a critical role in activating the transcription factor C/EBP-β-dependent gene expression in response to IFN-γ
Authors: Roy Sanjit K. , Hu Junbo , Meng Qingjun , Xia Ying , Shapiro Paul S. , Reddy Sekhar P. M. , Platanias Leonidas C. , Lindner Daniel J. , Johnson Peter F. , Pritchard Catrin , Pagés Gilles , Pouyssegur Jacques , Kalvakolanu Dhananjaya V.
Published: July 2002

IFN-γ induces a number of genes to up-regulate cellular responses by using specific transcription factors and the cognate elements. We recently discovered that CCAAT/enhancer-binding protein-β (C/EBP-β) induces gene transcription through an IFN-response element called γ-IFN-activated transcriptional element (GATE). Using mutant cells, chemical inhibitors, and specific dominant negative inhibitors, we show that induction of GATE-driven gene expression depends on MEK1 (mitogen-activated protein kinase kinase/extracellular signal-regulated protein kinase kinase) and ERKs (extracellular signal-regulated protein kinases) but is independent of Raf-1. Interestingly in cells lacking theMEKK1gene or expressing the dominant negative MEKK1, ERK activation, and GATE dependent gene expression is inhibited. A dominant negative MEKK1 blocks C/EBP-β-driven gene expression stimulated by IFN-γ. These studies describe an IFN-γ-stimulated pathway that involves MEKK1-MEK1-ERK1/2 kinases to regulate C/EBP-β-dependent gene expression.
Transduction of human NOD/SCID-repopulating cells with both lymphoid and myeloid potential by foamy virus vectors
Authors: Josephson Neil C. , Vassilopoulos George , Trobridge Grant D. , Priestley Greg V. , Wood Brent L. , Papayannopoulou Thalia , Russell David W.
Published: July 2002

The efficiency of gene transfer into human hematopoietic stem cells by oncoretroviral vectors is too low for effective gene therapy of most hematologic diseases. Retroviral vectors based on the nonpathogenic foamy viruses (FV) are an alternative gene-transfer system. In this study, human umbilical cord blood CD34
+
cells were transduced with FV vectors by a single 10-h exposure to vector stocks and then injected into sublethally irradiated nonobese diabetic-severe combined immunodeficiency (NOD/SCID) mice. At 5–7 weeks after transplantation, high transgene expression rates were observed in engrafted human hematopoietic cells, including over 60% of clonogenic progenitors. Significant transgene silencing did not occur. We developed an approach for expanding human cell populations derived from transplanted mice to show that multiple SCID repopulating cells (SRCs) had been transduced, including some that were capable of both lymphoid and myeloid differentiation. These findings demonstrate for the first time that human pluripotent (lympho-myeloid) hematopoietic stem cells repopulate NOD/SCID mice and can be efficiently transduced by FV vectors.
Using O
2
to probe membrane immersion depth by
19
F NMR
Authors: Prosser R. Scott , Luchette Paul A. , Westerman Philip W.
Published: July 2002

A fluorinated detergent, CF
3
(CF
2
)
5
C
2
H
4
-O-maltose, was reconstituted into a lipid bilayer model membrane system to demonstrate the feasibility of determining solvent accessibility and membrane immersion depth of each fluorinated group by
19
F NMR. Apolar oxygen, which is known to partition with an increasing concentration gradient toward the hydrophobic membrane interior, exhibits a range of paramagnetic relaxation effects on
19
F nuclei, depending on its depth in the membrane. This effect, which is predominately associated with spin-lattice relaxation rates (
R
1
) and chemical shifts, can be amplified greatly with minimal line broadening by increasing the partial pressure of O
2
at least 100-fold (i.e., P

O
2

greater than 20 bar). The differences of longitudinal relaxation rates at 20 bar of oxygen pressure to those under ambient pressure (
R
1
20bar
−
R
1
0
) are largest for those fluorine groups expected to be most deeply buried in the membrane bilayer. This result contrasts with the reverse trend, which is observed on addition of a membrane surface-associated paramagnetic species, 4-(
N
,
N
-dimethyl-
N
-hexadecyl) ammonium-2,2,6,6-tetramethylpiperidine-1-oxyl iodide (CAT-16) at ambient pressures. Thus, differential relaxation rates may be observed in
19
F-labeled membrane-associated molecules resulting from the addition of apolar oxygen under high pressure. The results demonstrate that the degree of solvent accessibility and membrane immersion depth of specific fluorinated species in membrane-associated macromolecules can be probed by
19
F NMR.
Suppression of Ras-mediated tumorigenicity and metastasis through inhibition of the Met receptor tyrosine kinase
Authors: Furge Kyle A. , Kiewlich David , Le Phuong , Vo My Nga , Faure Michel , Howlett Anthony R. , Lipson Kenneth E. , Woude George F. Vande , Webb Craig P.
Published: July 2002

Mutations in the Ras family of GTP binding proteins represent one of the most frequently observed genetic alterations in human cancers. We and others have recently demonstrated that expression of Met, the tyrosine kinase receptor for hepatocyte growth factor/scatter factor (HGF/SF), is significantly up-regulated in Ras-transformed cells. Because HGF/SF-Met signaling is proposed to play a prominent role in tumor development and progression, we assessed the possible requirement for Met during Ras-mediated tumor growth and metastasis. To disrupt endogenous Met signaling, we constructed dominant-negative mutants of both human and murine Met and showed that these can inhibit HGF/SF-mediated Met signaling and cell invasion of
ras
-transformed cells
in vitro
. Moreover, ectopic expression of dominant-negative Met mutants reduced the s.c. tumor growth of
ras
-transformed cells and dramatically suppressed their ability to form lung metastases
in vivo
. Our data demonstrate that Met plays a prominent role during Ras-mediated tumor growth and metastasis, and further suggest that agents that inhibit HGF/SF-Met signaling may represent an important therapeutic avenue for the treatment of a variety of malignant tumors.
The infiltration kinetics of simian immunodeficiency virus-specific T cells drawn to sites of high antigenic stimulation determines localin vivoviral escape
Authors: Blancou Philippe , Chenciner Nicole , Cumont Marie-Christine , Wain-Hobson Simon , Hurtrel Bruno , Cheynier Rémi
Published: July 2002

Despite vigorous cell-mediated immune responses to human and simian immunodeficiency viruses (HIV/SIV) the immune system is unable to clear latently infected resting T cells. These infected cells are reactivated by antigenic stimulation, leading to viral replication. By using the SIV/macaque model of HIV pathogenesis, the dynamics of T cell infiltration into delayed type hypersensitivity sites specific for the purified protein derivative of bacillus Calmette–Guérin have been studied. Early viral mRNA synthesis coincided with the infiltration of antigen-specific T cells. When the infiltration of anti-SIV-specific T cells was rapid compared with the kinetics of viral assembly, the sites were sterilized before the transition to late viral mRNA synthesis occurred. When their infiltration was slow, ephemeral foci of replication were identified. These findings were paralleled by plasma viremia; low viremia coincided with rapid sterilization of the delayed type hypersensitivity sites, whereas high load was found in association with local replication and delayed sterilization. These data suggest that although effective local control of SIV is possible once antiviral T lymphocytes have arrived on site, the slower deployment of these T cells may allow the virus to escape and thus to reseed the pool of memory T cells.
Regulation of cyclic peptide biosynthesis in a plant pathogenic fungus by a novel transcription factor
Authors: Pedley Kerry F. , Walton Jonathan D.
Published: July 2002

Strains of the filamentous fungus
Cochliobolus carbonum
that produce the host-selective compound HC-toxin, a cyclic tetrapeptide, are highly virulent on certain genotypes of maize (
Zea mays
L.). Production of HC-toxin is under the control of a complex locus,
TOX2
, which is composed of at least seven linked and duplicated genes that are present only in toxin-producing strains of
C. carbonum
. One of these genes,
TOXE
, was earlier shown to be required for the expression of the other
TOX2
genes. TOXE has four ankyrin repeats and a basic region similar to those found in basic leucine zipper (bZIP) proteins, but lacks any apparent leucine zipper. Here we show that TOXE is a DNA-binding protein that recognizes a ten-base motif (the “tox-box”) without dyad symmetry that is present in the promoters of all of the known
TOX2
genes. Both the basic region and the ankyrin repeats are involved in DNA binding. A region of TOXE that includes the first ankyrin repeat is necessary and sufficient for transcriptional activation in yeast. The data indicate that TOXE is the prototype of a new family of transcription factor, so far found only in plant-pathogenic fungi. TOXE plays a specific regulatory role in HC-toxin production and, therefore, pathogenicity by
C. carbonum
.
Members of the HRT family of basic helix–loop–helix proteins act as transcriptional repressors downstream of Notch signaling
Authors: Nakagawa Osamu , McFadden David G. , Nakagawa Masayo , Yanagisawa Hiromi , Hu Tonghuan , Srivastava Deepak , Olson Eric N.
Published: July 2002

The
Hairy-related transcription-factor
(
HRT
) genes encode three related basic
helix–loop–helix transcription factors that show sequence similarity
to the Hairy and Enhancer of split family of transcriptional
repressors. HRT proteins are expressed in specific regions of the
developing heart, vasculature, pharyngeal arches and somites, and the
periodicity of their expression in somitic precursors mirrors that of
Notch signaling-related molecules. In the present study, we show that
the intracellular domain of the Notch1 receptor (Notch1 IC), which is
constitutively active, up-regulates
HRT2
expression in
10T½ fibroblasts. Luciferase reporter assays using the
regulatory regions of the mouse
HRT
genes revealed that
transcription of all three genes is stimulated by Notch1 IC. The
promoters of the
HRT
genes share homology in a binding
site for Suppressor of Hairless [Su(H)], a transcriptional mediator
of Notch signaling. A dominant-negative Su(H) mutant abolished
Notch-activated
HRT2
expression, and mutation of the
conserved Su(H) consensus site in the
HRT2
promoter
attenuated transcriptional activation by Notch. Ectopic expression of
HRT proteins also blocked activation of
HRT2
expression
by Notch1 IC through a mechanism requiring the basic region, but not
the conserved carboxyl-terminal YQPW-TEVGAF motif of HRT2. These
findings identify
HRT
genes as downstream targets for
Notch signaling and reveal a negative autoregulatory loop whereby HRT
proteins repress their own expression through interference with Notch
signaling.
A peptide derived from α-fetoprotein prevents the growth of estrogen-dependent human breast cancers sensitive and resistant to tamoxifen
Authors: Bennett James A. , Mesfin Fassil B. , Andersen Thomas T. , Gierthy John F. , Jacobson Herbert I.
Published: July 2002

An 8-mer peptide (EMTOVNOG) derived from α-fetoprotein was compared with tamoxifen for activity against growth of human breast cancer xenografts implanted in immune-deficient mice. Both peptide and tamoxifen prevented growth of estrogen-receptor-positive MCF-7 and T47D human breast cancer xenografts. A subline of MCF-7, made resistant to tamoxifen by a 6-month exposure to this drug in culture, was found to be resistant to tamoxifen
in vivo
. Peptide completely prevented the xenograft growth of this tamoxifen-resistant subline of MCF-7. Neither peptide nor tamoxifen was effective in slowing the xenograft growth of the estrogen-receptor-negative MDA-MB-231 human breast cancer. A worrisome side effect of tamoxifen is its hypertrophic effect on the uterus. In this study, tamoxifen was shown to stimulate the growth of the immature mouse uterus
in vivo
, and the peptide significantly inhibited tamoxifen's uterotrophic effect. The mechanism of action of peptide is different from that of tamoxifen in that the peptide does not interfere with the binding of [
3
H]estradiol to the estrogen receptor. In conclusion, α-fetoprotein-derived peptide appears to be a novel agent that interferes with the growth of tamoxifen-sensitive as well as tamoxifen-resistant estrogen-receptor-positive human breast cancers; it inhibits the uterotrophic side effect of tamoxifen and, thus, it may be useful in combination with or in place of tamoxifen for treatment of estrogen-receptor-positive human breast cancers.
Molecular mechanisms of dexamethasone inhibition of nitric oxide synthase expression in interleukin 1 beta-stimulated mesangial cells: evidence for the involvement of transcriptional and posttranscriptional regulation.
Authors: Kunz D , Walker G , Eberhardt W , Pfeilschifter J
Published: July 2002

Inducible nitric oxide synthase (iNOS; EC 1.14.13.39) is expressed in rat glomerular mesangial cells upon exposure to the inflammatory cytokine interleukin 1 beta (IL-1 beta). We have reported that nanomolar concentrations of dexamethasone suppress IL-1 beta-induced iNOS protein expression and production of nitrite, the stable end product of NO formation, without affecting IL-1 beta-triggered increase in iNOS mRNA levels. We now have studied the mechanisms by which dexamethasone suppresses IL-1 beta-stimulated iNOS expression in mesangial cells. Surprisingly, nuclear run-on transcription experiments demonstrate that dexamethasone markedly attenuates IL-1 beta-induced iNOS gene transcription. However, this is counteracted by a prolongation of the half-life of iNOS mRNA from 1 h to 2.5 h by dexamethasone. Moreover, dexamethasone drastically reduces the amount of iNOS protein by reduction of iNOS mRNA translation and increased degradation of iNOS protein. These results indicate that glucocorticoids act at multiple levels to regulate iNOS expression, thus providing important insights into the treatment of inflammatory diseases.
Cells expressing the DG42 gene from early Xenopus embryos synthesize hyaluronan.
Authors: Meyer M F , Kreil G
Published: July 2002

DG42 is one of the main mRNAs expressed during gastrulation in embryos of Xenopus laevis. Here we demonstrate that cells expressing this mRNA synthesize hyaluronan. The cloned DG42 cDNA was expressed in rabbit kidney (RK13) and human osteosarcoma (tk-) cells using a vaccinia virus system. Lysates prepared from infected cells were incubated in the presence of UDP-N-acetylglucosamine and UDP-[14C]glucuronic acid. This yielded a glycosaminoglycan with a molecular mass of about 200,000 Da. Formation of this product was only observed in the presence of both substrates. The glycosaminoglycan could be digested with testicular hyaluronidase and with Streptomyces hyaluronate lyase but not with Serratia chitinase. Hyaluronan synthase activity could also be detected in homogenates of early Xenopus embryos, and the activity was found to correlate with the expression of DG42 mRNA at different stages of development. Synthesis of hyaluronan is thus an early event after midblastula transition, indicating its importance for the ensuing cell movements in the developing embryo. Our results are at variance with a recent report (Semino, C. E. & Robbins, P. W. (1995) Proc. Natl. Acad. Sci. USA 92, 3498-3501) that DG42 codes for an enzyme that catalyzes the synthesis of chitin-like oligosaccharides.
Homologs of the Xenopus developmental gene DG42 are present in zebrafish and mouse and are involved in the synthesis of Nod-like chitin oligosaccharides during early embryogenesis.
Authors: Semino C E , Specht C A , Raimondi A , Robbins P W
Published: July 2002

The Xenopus developmental gene DG42 is expressed during early embryonic development, between the midblastula and neurulation stages. The deduced protein sequence of Xenopus DG42 shows similarity to Rhizobium Nod C, Streptococcus Has A, and fungal chitin synthases. Previously, we found that the DG42 protein made in an in vitro transcription/translation system catalyzed synthesis of an array of chitin oligosaccharides. Here we show that cell extracts from early Xenopus and zebrafish embryos also synthesize chitooligosaccharides. cDNA fragments homologous to DG42 from zebrafish and mouse were also cloned and sequenced. Expression of these homologs was similar to that described for Xenopus based on Northern and Western blot analysis. The Xenopus anti-DG42 antibody recognized a 63-kDa protein in extracts from zebrafish embryos that followed a similar developmental expression pattern to that previously described for Xenopus. The chitin oligosaccharide synthase activity found in extracts was inactivated by a specific DG42 antibody; synthesis of hyaluronic acid (HA) was not affected under the conditions tested. Other experiments demonstrate that expression of DG42 under plasmid control in mouse 3T3 cells gives rise to chitooligosaccharide synthase activity without an increase in HA synthase level. A possible relationship between our results and those of other investigators, which show stimulation of HA synthesis by DG42 in mammalian cell culture systems, is provided by structural analyses to be published elsewhere that suggest that chitin oligosaccharides are present at the reducing ends of HA chains. Since in at least one vertebrate system hyaluronic acid formation can be inhibited by a pure chitinase, it seems possible that chitin oligosaccharides serve as primers for hyaluronic acid synthesis.
Limited surface exposure of Borrelia burgdorferi outer surface lipoproteins.
Authors: Cox D L , Akins D R , Bourell K W , Lahdenne P , Norgard M V , Radolf J D
Published: July 2002

We used novel immunofluorescence strategies to demonstrate that outer surface proteins (Osps) A, B and C of Borrelia burgdorferi have limited surface exposure, finding that contradicts the prevailing viewpoint that these antigens are exclusively surface exposed. Light labeling was observed when antibodies to OspA or OspB were added to motile organisms, whereas intense fluorescence was observed when the same slides were methanol-fixed and reprobed. Modest labeling also was observed when spirochetes encapsulated in agarose beads (gel microdroplets) were incubated with antibodies to these same two antigens. This contrasted with the intense fluorescence observed when encapsulated spirochetes were probed in the presence of 0.06% Triton X-100, which selectively removed outer membranes. Proteinase K (PK) treatment of encapsulated spirochetes abrogated surface labeling. However, PK-treated spirochetes fluoresced intensely after incubation with antibodies to OspA or OspB in the presence of detergent, confirming the existence of large amounts of subsurface Osp antigens. Modest surface labeling once again was detected when PK-treated spirochetes were reprobed after overnight incubation, a result consistent with the existence of a postulated secretory apparatus that shuttles lipoproteins to the borrelial surface. Last, experiments with the OspC-expressing B. burgdorferi strain 297 revealed that this antigen was barely detectable on spirochetal surfaces even though it was a major constituent of isolated outer mem- branes. We propose a model of B. burgdorferi molecular architecture that helps to explain spirochetal persistence during chronic Lyme disease.
Interaction between human tRNA synthetases involves repeated sequence elements.
Authors: Rho S B , Lee K H , Kim J W , Shiba K , Jo Y J , Kim S
Published: July 2002

Aminoacyl-tRNA synthetases (tRNA synthetases) of higher eukaryotes form a multiprotein complex. Sequence elements that are responsible for the protein assembly were searched by using a yeast two-hybrid system. Human cytoplasmic isoleucyl-tRNA synthetase is a component of the multi-tRNA synthetase complex and it contains a unique C-terminal appendix. This part of the protein was used as bait to identify an interacting protein from a HeLa cDNA library. The selected sequence represented the internal 317 amino acids of human bifunctional (glutamyl- and prolyl-) tRNA synthetase, which is also known to be a component of the complex. Both the C-terminal appendix of the isoleucyl-tRNA synthetase and the internal region of bifunctional tRNA synthetase comprise repeating sequence units, two repeats of about 90 amino acids, and three repeats of 57 amino acids, respectively. Each repeated motif of the two proteins was responsible for the interaction, but the stronger interaction was shown by the native structures containing multiple motifs. Interestingly, the N-terminal extension of human glycyl-tRNA synthetase containing a single motif homologous to those in the bifunctional tRNA synthetase also interacted with the C-terminal motif of the isoleucyl-tRNA synthetase although the enzyme is not a component of the complex. The data indicate that the multiplicity of the binding motif in the tRNA synthetases is necessary for enhancing the interaction strength and may be one of the determining factors for the tRNA synthetases to be involved in the formation of the multi-tRNA synthetase complex.
Sequence-selective carbohydrate-DNA interaction: dimeric and monomeric forms of the calicheamicin oligosaccharide interfere with transcription factor function.
Authors: Liu C , Smith B M , Ajito K , Komatsu H , Gomez-Paloma L , Li T , Theodorakis E A , Nicolaou K C , Vogt P K
Published: July 2002

The synthetic oligosaccharide moiety of the antibiotic calicheamicin and the head-to-head dimer of this oligosaccharide are known to bind to the minor groove of DNA in a sequence-selective manner preferring distinct target sequences. We tested these carbohydrates for their ability to interfere with transcription factor function. The oligosaccharides inhibit binding of transcription factors to DNA in a sequence-selective manner, probably by inducing a conformational change in DNA structure. They also interfere with transcription by polymerase II in vitro. The effective concentrations of the oligosaccharides for inhibition of transcription factor binding and for transcriptional inhibition are in the micromolar range. The dimer is a significantly more active inhibitor than is the monomer.
Vaccination with syngeneic, lymphoma-derived immunoglobulin idiotype combined with granulocyte/macrophage colony-stimulating factor primes mice for a protective T-cell response.
Authors: Kwak L W , Young H A , Pennington R W , Weeks S D
Published: July 2002

The idiotype of the Ig expressed by a B-cell malignancy (Id) can serve as a unique tumor-specific antigen and as a model for cancer vaccine development. In murine models of Id vaccination, formulation of syngeneic Id with carrier proteins or adjuvants induces an anti-idiotypic antibody response. However, inducing a potent cell-mediated response to this weak antigen instead would be highly desirable. In the 38C13 lymphoma model, we observed that low doses of free granulocyte/macrophage colony-stimulating factor (GM-CSF) 10,000 units i.p. or locally s.c. daily for 4 days significantly enhanced protective antitumor immunity induced by s.c. Id-keyhole limpet hemocyanin (KLH) immunization. This effect was critically dependent upon effector CD4+ and CD8+ T cells and was not associated with any increased anti-idiotypic antibody production. Lymphocytes from spleens and draining lymph nodes of mice primed with Id-KLH plus GM-CSF, but not with Id-KLH alone, demonstrated significant proliferation to Id in vitro without any biased production of interferon gamma or interleukin 4 protein or mRNA. As a further demonstration of potency, 50% of mice immunized with Id-KLH plus GM-CSF on the same day as challenge with a large s.c. tumor inoculum remained tumor-free at day 80, compared with 17% for Id-KLH alone, when immunization was combined with cyclophosphamide. Taken together, these results demonstrate that GM-CSF can significantly enhance the immunogenicity of a defined self-antigen and that this effect is mediated exclusively by activating the T-cell arm of the immune response.
Molecular clock or erratic evolution? A tale of two genes.
Authors: Ayala F J , Barrio E , Kwiatowski J
Published: July 2002

We have investigated the evolution of glycerol-3-phosphate dehydrogenase (Gpdh). The rate of amino acid replacements is 1 x 10(-10)/site/year when Drosophila species are compared. The rate is 2.7 times greater when Drosophila and Chymomyza species are compared; and about 5 times greater when any of those species are compared with the medfly Ceratitis capitata. This rate of 5 x 10(-10)/site/year is also the rate observed in comparisons between mammals, or between different animal phyla, or between the three multicellular kingdoms. We have also studied the evolution of Cu,Zn superoxide dismutase (Sod). The rate of amino acid replacements is about 17 x 10(-10)/site/year when comparisons are made between dipterans or between mammals, but only 5 x 10(-10) when animal phyla are compared, and only 3 x 10(-10) when the multicellular kingdoms are compared. The apparent decrease by about a factor of 5 in the rate of SOD evolution as the divergence between species increases can be consistent with the molecular clock hypothesis by assuming the covarion hypothesis (namely, that the number of amino acids that can change is constant, but the set of such amino acids changes from time to time and from lineage to lineage). However, we know of no model consistent with the molecular clock hypothesis that would account for the increase in the rate of GPDH evolution as the divergence between species increases.
Are the returns to technological change in health care declining?
Authors: McClellan Mark
Published: July 2002

Whether the U.S. health care system supports too much technological
change—so that new technologies of low value are adopted, or
worthwhile technologies become overused—is a controversial question.
This paper analyzes the marginal value of technological change for
elderly heart attack patients in 1984–1990. It estimates the
additional benefits and costs of treatment by hospitals that are likely
to adopt new technologies first or use them most intensively. If the
overall value of the additional treatments is declining, then the
benefits of treatment by such intensive hospitals relative to other
hospitals should decline, and the additional costs of treatment by such
hospitals should rise. To account for unmeasured changes in patient mix
across hospitals that might bias the results, instrumental–variables
methods are used to estimate the incremental mortality benefits and
costs. The results do not support the view that the returns to
technological change are declining. However, the incremental value of
treatment by intensive hospitals is low throughout the study period,
supporting the view that new technologies are overused.
Regulation of transforming growth factor β- and activin-induced transcription by mammalian Mad proteins
Authors: Chen Yan , Lebrun Jean-Jacques , Vale Wylie
Published: July 2002

Members of the transforming growth factor β (TGF-β) superfamily are involved in diverse physiological activities including development, tissue repair, hormone regulation, bone formation, cell growth, and differentiation. At the cellular level, these functions are initiated by the interaction of ligands with specific transmembrane receptors with intrinsic serine/threonine kinase activity. The signaling pathway that links receptor activation to the transcriptional regulation of the target genes is largely unknown. Recent work inDrosophilaandXenopussignaling suggested that Mad (Mothers againstdpp) functions downstream of the receptors of the TGF-β family. Mammalian Mad1 has been reported to respond to bone morphogenetic protein (BMP), but not to TGF-β or activin. We report here the cloning and functional studies of a novel mammalian Mad molecule, Mad3, as well as a rat Mad1 homologue. Overexpression of Mad3 in a variety of cells stimulated basal transcriptional activity of the TGF-β/activin-responsive reporter construct, p3TP-Lux. Furthermore, expression of Mad3 could potentiate the TGF-β- and activin-induced transcriptional stimulation of p3TP-Lux. By contrast, overexpression of Mad1 inhibited the basal as well as the TGF-β/activin induced p3TP-Lux activity. These findings, therefore, support the hypothesis that Mad3 may serve as a mediator linking TGF-β/activin receptors to transcriptional regulation.
Integration of
Agrobacterium tumefaciens
T-DNA in the
Saccharomyces cerevisiae
genome by illegitimate recombination
Authors: Bundock Paul , Hooykaas Paul J. J.
Published: July 2002

Agrobacterium tumefaciens
can transfer part of its
Ti plasmid, the T-DNA, to plant cells where it integrates into the
nuclear genome via illegitimate recombination. Integration of the T-DNA
results in small deletions of the plant target DNA, and may lead to
truncation of the T-DNA borders and the production of filler DNA. We
showed previously that T-DNA can also be transferred from
A.
tumefaciens
to
Saccharomyces cerevisiae
and
integrates into the yeast genome via homologous recombination. We show
here that when the T-DNA lacks homology with the
S.
cerevisiae
genome, it integrates at random positions via
illegitimate recombination. From 11 lines the integrated T-DNA was
cloned back to
Escherichia coli
along with yeast
flanking sequences. The T-DNA borders and yeast DNA flanking the T-DNA
were sequenced and characterized. It was found that T-DNA integration
had resulted in target DNA deletions and sometimes T-DNA truncations or
filler DNA formation. Therefore, the molecular mechanism of
illegitimate recombination by which T-DNA integrates in higher and
lower eukaryotes seems conserved.
Impaired hippocampal plasticity in mice lacking the Cbeta1 catalytic subunit of cAMP-dependent protein kinase.
Authors: Qi M , Zhuo M , Skålhegg B S , Brandon E P , Kandel E R , McKnight G S , Idzerda R L
Published: July 2002

Neural pathways within the hippocampus undergo use-dependent changes in synaptic efficacy, and these changes are mediated by a number of signaling mechanisms, including cAMP-dependent protein kinase (PKA). The PKA holoenzyme is composed of regulatory and catalytic (C) subunits, both of which exist as multiple isoforms. There are two C subunit genes in mice, Calpha and Cbeta, and the Cbeta gene gives rise to several splice variants that are specifically expressed in discrete regions of the brain. We have used homologous recombination in embryonic stem cells to introduce an inactivating mutation into the mouse Cbeta gene, specifically targeting the Cbeta1-subunit isoform. Homozygous mutants showed normal viability and no obvious pathological defects, despite a complete lack of Cbeta1. The mice were analyzed in electrophysiological paradigms to test the role of this isoform in long-term modulation of synaptic transmission in the Schaffer collateral-CA1 pathway of the hippocampus. A high-frequency stimulus produced potentiation in both wild-type and Cbeta1-/- mice, but the mutants were unable to maintain the potentiated response, resulting in a late phase of long-term potentiation that was only 30% of controls. Paired pulse facilitation was unaffected in the mutant mice. Low-frequency stimulation produced long-term depression and depotentiation in wild-type mice but failed to produce lasting synaptic depression in the Cbeta1 -/- mutants. These data provide direct genetic evidence that PKA, and more specifically the Cbeta1 isoform, is required for long-term depression and depotentiation, as well as the late phase of long-term potentiation in the Schaffer collateral-CA1 pathway.
Targeted gene expression without a tissue-specific promoter: Creating mosaic embryos using laser-induced single-cell heat shock
Authors: Halfon Marc S. , Kose Hiroyuki , Chiba Akira , Keshishian Haig
Published: July 2002

We have developed a method to target gene expression in theDrosophilaembryo to a specific cell without having a promoter that directs expression in that particular cell. Using a digitally enhanced imaging system to identify single cells within the living embryo, we apply a heat shock to each cell individually by using a laser microbeam. A 1- to 2-min laser treatment is sufficient to induce a heat-shock response but is not lethal to the heat-shocked cells. Induction of heat shock was measured in a variety of cell types, including neurons and somatic muscles, by the expression of β-galactosidase from anhsp26-lacZreporter construct or by expression of a UAS target gene after induction ofhsGAL4. We discuss the applicability of this technique to ectopic gene expression studies, lineage tracing, gene inactivation studies, and studies of cellsin vitro. Laser heat shock is a versatile technique that can be adapted for use in a variety of research organisms and is useful for any studies in which it is desirable to express a given gene in only a distinct cell or clone of cells, either transiently or constitutively, at a time point of choice.
In vivo
activity-dependent plasticity at cortico-striatal connections: Evidence for physiological long-term potentiation
Authors: Charpier S. , Deniau J. M.
Published: July 2002

The purpose of the present study was to investigate
in vivo
the activity-dependent plasticity of glutamatergic cortico-striatal synapses. Electrical stimuli were applied in the facial motor cortex and intracellular recordings were performed in the ipsilateral striatal projection field of this cortical area. Recorded cells exhibited the typical intrinsic membrane properties of striatal output neurons and were identified morphologically as medium spiny type I neurons. Subthreshold cortical tetanization produced either short-term posttetanic potentiation or short-term depression of cortically-evoked excitatory postsynaptic potentials. When coupled with a postsynaptic depolarization leading the membrane potential to a suprathreshold level, the tetanus induced long-term potentiation (LTP) of cortico-striatal synaptic transmission. Induction of striatal LTP was prevented by intracellular injection of a calcium chelator suggesting that this synaptic plasticity involves an increase of postsynaptic free calcium concentration. Contrasting with previous
in vitro
studies our findings demonstrate that LTP constitutes the normal form of use-dependent plasticity at cortico-striatal synapses. Since excitation of striatal neurons produces a disinhibition of premotor networks, LTP at excitatory striatal inputs should favor the initiation of movements and therefore could be critical for the functions of basal ganglia in motor learning.
Structural protein 4.1 is located in mammalian centrosomes
Authors: Krauss Sharon Wald , Chasis Joel Anne , Rogers Catherine , Mohandas Narla , Krockmalnic Gabriela , Penman Sheldon
Published: July 2002

Structural protein 4.1 was first characterized as an important 80-kDa protein in the mature red cell membrane skeleton. It is now known to be a member of a family of protein isoforms detected at diverse intracellular sites in many nucleated mammalian cells. We recently reported that protein 4.1 isoforms are present at interphase in nuclear matrix and are rearranged during the cell cycle. Here we report that protein 4.1 epitopes are present in centrosomes of human and murine cells and are detected by using affinity-purified antibodies specific for 80-kDa red cell 4.1 and for 4.1 peptides. Immunofluorescence, by both conventional and confocal microscopy, showed that protein 4.1 epitopes localized in the pericentriolar region. Protein 4.1 epitopes remained in centrosomes after extraction of cells with detergent, salt, and DNase. Higher resolution electron microscopy of detergent-extracted cell whole mounts showed centrosomal protein 4.1 epitopes distributed along centriolar cylinders and on pericentriolar fibers, at least some of which constitute the filamentous network surrounding each centriole. Double-label electron microscopy showed that protein 4.1 epitopes were predominately localized in regions also occupied by epitopes for centrosome-specific autoimmune serum 5051 but were not found on microtubules. Our results suggest that protein 4.1 is an integral component of centrosome structure, in which it may play an important role in centrosome function during cell division and organization of cellular architecture.