bluuebunny/crossref_metadata_2025 huggingface dataset:
Jun NH2-terminal kinase is constitutively activated in T cells transformed by the intracellular parasiteTheileria parva
Authors: Galley Yves , Hagens Gerry , Glaser Isabel , Davis William , Eichhorn Margarete , Dobbelaere Dirk
Published: July 2002

When T cells become infected by the parasiteTheileria parva,they acquire a transformed phenotype and no longer require antigen-specific stimulation or exogenous growth factors. This is accompanied by constitutive interleukin 2 (IL-2) and IL-2 receptor expression. Transformation can be reversed entirely by elimination of the parasites using the specific drug BW720c. Extracellular signal-regulated kinase and jun NH2-terminal kinase (JNK) are members of the mitogen-activated protein kinase family, which play a central role in the regulation of cellular differentiation and proliferation and also participate in the regulation of IL-2 and IL-2 receptor gene expression.T. parvawas found to induce an unorthodox pattern of mitogen-activated protein kinase expression in infected T cells. JNK-1 and JNK-2 are constitutively active in a parasite-dependent manner, but have altered properties. In contrast, extracellular signal-regulated kinase-2 is not activated even though its activation pathway is functionally intact. Different components of the T cell receptor (TCR)-dependent signal transduction pathways also were examined. The TCRζ or CD3ɛ chains were found not to be phosphorylated andT. parva-transformed T cells were resistant to inhibitors that block the early steps of T cell activation. Compounds that inhibit the progression of T cells to proliferation, however, were inhibitory. Our data provide the first example, to our knowledge, for parasite-mediated JNK activation, and our findings strongly suggest thatT. parvanot only lifts the requirement for antigenic stimulation but also entirely bypasses early TCR-dependent signal transduction pathways to induce continuous proliferation.
Elevation of intracellular calcium in smooth muscle causes endothelial cell generation of NO in arterioles
Authors: Dora K. A. , Doyle M. P. , Duling B. R.
Published: July 2002

It is well known that vascular smooth muscle tone can be modulated by signals arising in the endothelium (e.g., endothelium-derived relaxing factor, endothelium-derived hyperpolarizing factor, and prostaglandins). Here we show that during vasoconstriction a signal can originate in smooth muscle cells and act on the endothelium to cause synthesis of endothelium-derived relaxing factor. We studied responses to two vasoconstrictors (phenylephrine and KCl) that act by initiating a rise in smooth muscle cell intracellular Ca
2+
concentration ([Ca
2+
]
i
) and exert little or no direct effect on the endothelium. Fluo-3 was used as a Ca
2+
indicator in either smooth muscle or endothelial cells of arterioles from the hamster cheek pouch. Phenylephrine and KCl caused the expected rise in smooth muscle cell [Ca
2+
]
i
that was accompanied by an elevation in endothelial cell [Ca
2+
]
i
. The rise in endothelial cell [Ca
2+
]
i
was followed by increased synthesis of NO, as evidenced by an enhancement of the vasoconstriction induced by both agents after blockade of NO synthesis. The molecule involved in signal transmission from smooth muscle to endothelium is as yet unknown. However, given that myoendothelial cell junctions are frequent in these vessels, we hypothesize that the rise in smooth muscle cell Ca
2+
generates a diffusion gradient that drives Ca
2+
through myoendothelial cell junctions and into the endothelial cells, thereby initiating the synthesis of NO.
Vascular endothelial growth factor: Crystal structure and functional mapping of the kinase domain receptor binding site
Authors: Muller Yves A. , Li Bing , Christinger Hans W. , Wells James A. , Cunningham Brian C. , de Vos Abraham M.
Published: July 2002

Vascular endothelial growth factor (VEGF) is a homodimeric member of the cystine knot family of growth factors, with limited sequence homology to platelet-derived growth factor (PDGF) and transforming growth factor β2 (TGF-β). We have determined its crystal structure at a resolution of 2.5 Å, and identified its kinase domain receptor (KDR) binding site using mutational analysis. Overall, the VEGF monomer resembles that of PDGF, but its N-terminal segment is helical rather than extended. The dimerization mode of VEGF is similar to that of PDGF and very different from that of TGF-β. Mutational analysis of VEGF reveals that symmetrical binding sites for KDR are located at each pole of the VEGF homodimer. Each site contains two functional “hot spots” composed of binding determinants presented across the subunit interface. The two most important determinants are located within the largest hot spot on a short, three-stranded sheet that is conserved in PDGF and TGF-β. Functional analysis of the binding epitopes for two receptor-blocking antibodies reveal different binding determinants near each of the KDR binding hot spots.
Genetics and the origin of bird species
Authors: Grant Peter R. , Grant B. Rosemary
Published: July 2002

External (environmental) factors affecting the speciation of birds are better known than the internal (genetic) factors. The opposite is true for several groups of invertebrates,Drosophilabeing the outstanding example. Ideas about the genetics of speciation in general trace back to Dobzhansky who worked withDrosophila. These ideas are an insufficient guide for reconstructing speciation in birds for two main reasons. First, speciation in birds proceeds with the evolution of behavioral barriers to interbreeding; postmating isolation usually evolves much later, perhaps after gene exchange has all but ceased. As a consequence of the slow evolution of postmating isolating factors the scope for reinforcement of premating isolation is small, whereas the opportunity for introgressive hybridization to influence the evolution of diverging species is large. Second, premating isolation may arise from nongenetic, cultural causes; isolation may be affected partly by song, a trait that is culturally inherited through an imprinting-like process in many, but not all, groups of birds. Thus the genetic basis to the origin of bird species is to be sought in the inheritance of adult traits that are subject to natural and sexual selection. Some of the factors involved in premating isolation (plumage, morphology, and behavior) are under single-gene control, most are under polygenic control. The genetic basis of the origin of postmating isolating factors affecting the early development of embryos (viability) and reproductive physiology (sterility) is almost completely unknown. Bird speciation is facilitated by small population size, involves few genetic changes, and occurs relatively rapidly.
CCAAT/enhancer binding protein (C/EBP) sites are required for HIV-1 replication in primary macrophages but not CD4+T cells
Authors: Henderson A. J. , Calame K. L.
Published: July 2002

The importance of CCAAT/enhancer binding proteins (C/EBPs) and binding sites for HIV-1 replication in primary macrophages, T cell lines and primary CD4+T cells was examined. When lines overexpressing the C/EBP dominant-negative protein LIP were infected with HIV-1, replication occurred in Jurkat T cells but not in U937 promonocytes, demonstrating a requirement for C/EBP activators by HIV-1 only in promonocytes. Primary macrophages did not support the replication of HIV-1 harboring mutant C/EBP binding sites in the long terminal repeat but Jurkat, H9 and primary CD4+T cells supported replication of wild-type and mutant HIV-1 equally well. Thus the requirement for C/EBP sites is also confined to monocyte/macrophages. The requirement for C/EBP proteins and sites identifies the first uniquely macrophage-specific regulatory mechanism for HIV-1 replication.
p53-independent apoptosis induced by paclitaxel through an indirect mechanism
Authors: Lanni Jennifer S. , Lowe Scott W. , Licitra Edward J. , Liu Jun O. , Jacks Tyler
Published: July 2002

The efficacy of chemotherapeutic agents may be determined by a number of different factors, including the genotype of the tumor cell. The p53 tumor suppressor gene frequently is mutated in human tumors, and this may contribute to chemotherapeutic resistance. We tested the requirement for wild-type p53 in the response of tumor cells to treatment with paclitaxel (trade name Taxol), an antineoplastic agent that stabilizes cellular microtubules. Although paclitaxel is broadly effective against human tumor xenografts in mice, including some known to carry p53 mutations, we found that p53-containing mouse tumor cells were significantly more sensitive to direct treatment with this drug than were p53-deficient tumor cells. In an attempt to reconcile this apparent discrepancy, we examined the requirement for p53 in the cytotoxic effects of tumor necrosis factor α (TNF-α), a cytokine released from murine macrophages upon paclitaxel treatment. Conditioned medium from paclitaxel-treated macrophages was capable of inducing p53-independent apoptosis when applied to transformed mouse embryonic fibroblasts and was inhibitable by antibodies against TNF-α. Furthermore, in response to direct treatment with TNF-α, both wild-type and p53-deficient tumor cells underwent apoptosis to similar extents and with similar kinetics. Our results suggest that the efficacy of paclitaxelin vivomay be due not only to its microtubule-stabilizing activity, but its ability to activate local release of an apoptosis-inducing cytokine.
A mammalian model for Laron syndrome produced by targeted disruption of the mouse growth hormone receptor/binding protein gene (the Laron mouse)
Authors: Zhou Yihua , Xu Bixiong C. , Maheshwari Hiralal G. , He Li , Reed Michael , Lozykowski Maria , Okada Shigeru , Cataldo Lori , Coschigamo Karen , Wagner Thomas E. , Baumann Gerhard , Kopchick John J.
Published: July 2002

Laron syndrome [growth hormone (GH) insensitivity syndrome] is a hereditary dwarfism resulting from defects in the GH receptor (GHR) gene. GHR deficiency has not been reported in mammals other than humans. Many aspects of GHR dysfunction remain unknown because of ethical and practical limitations in studying humans. To create a mammalian model for this disease, we generated mice bearing a disrupted GHR/binding protein (GHR/BP) gene through a homologous gene targeting approach. Homozygous GHR/BP knockout mice showed severe postnatal growth retardation, proportionate dwarfism, absence of the GHR and GH binding protein, greatly decreased serum insulin-like growth factor I and elevated serum GH concentrations. These characteristics represent the phenotype typical of individuals with Laron syndrome. Animals heterozygous for the GHR/BP defect show only minimal growth impairment but have an intermediate biochemical phenotype, with decreased GHR and GH binding protein expression and slightly diminished insulin-like growth factor I levels. These findings indicate that the GHR/BP-deficient mouse (Laron mouse) is a suitable model for human Laron syndrome that will prove useful for the elucidation of many aspects of GHR/BP function that cannot be obtained in humans.
Catalytic mechanism of the adenylyl and guanylyl cyclases: Modeling and mutational analysis
Authors: Liu Yu , Ruoho Arnold E. , Rao Vibha D. , Hurley James H.
Published: July 2002

The adenylyl and guanylyl cyclases catalyze the formation of 3′,5′-cyclic adenosine or guanosine monophosphate from the corresponding nucleoside 5′-triphosphate. The guanylyl cyclases, the mammalian adenylyl cyclases, and their microbial homologues function as pairs of homologous catalytic domains. The crystal structure of the rat type II adenylyl cyclase C
2
catalytic domain was used to model by homology a mammalian adenylyl cyclase C
1
-C
2
domain pair, a homodimeric adenylyl cyclase of
Dictyostelium discoideum
, a heterodimeric soluble guanylyl cyclase, and a homodimeric membrane guanylyl cyclase. Mg
2+
ATP or Mg
2+
GTP were docked into the active sites based on known stereochemical constraints on their conformation. The models are consistent with the activities of seven active-site mutants. Asp-310 and Glu-432 of type I adenylyl cyclase coordinate a Mg
2+
ion. The D310S and D310A mutants have 10-fold reduced
V
max
and altered [Mg
2+
] dependence. The NTP purine moieties bind in mostly hydrophobic pockets. Specificity is conferred by a Lys and an Asp in adenylyl cyclase, and a Glu, an Arg, and a Cys in guanylyl cyclase. The models predict that an Asp from one domain is a general base in the reaction, and that the transition state is stabilized by a conserved Asn-Arg pair on the other domain.
Chaperone-supervised conversion of prion protein to its protease-resistant form
Authors: DebBurman Shubhik K. , Raymond Gregory J. , Caughey Byron , Lindquist Susan
Published: July 2002

Transmissible spongiform encephalopathies (TSEs) are lethal, infectious disorders of the mammalian nervous system. A TSE hallmark is the conversion of the cellular protein PrP
C
to disease-associated PrP
Sc
(named for scrapie, the first known TSE). PrP
C
is protease-sensitive, monomeric, detergent soluble, and primarily α-helical; PrP
Sc
is protease-resistant, polymerized, detergent insoluble, and rich in β-sheet. The “protein-only” hypothesis posits that PrP
Sc
is the infectious TSE agent that directly converts host-encoded PrP
C
to fresh PrP
Sc
, harming neurons and creating new agents of infection. To gain insight on the conformational transitions of PrP, we tested the ability of several protein chaperones, which supervise the conformational transitions of proteins in diverse ways, to affect conversion of PrP
C
to its protease-resistant state. None affected conversion in the absence of pre-existing PrP
Sc
. In its presence, only two, GroEL and Hsp104 (heat shock protein 104), significantly affected conversion. Both promoted it, but the reaction characteristics of conversions with the two chaperones were distinct. In contrast, chemical chaperones inhibited conversion. Our findings provide new mechanistic insights into nature of PrP conversions, and provide a new set of tools for studying the process underlying TSE pathogenesis.
Recovery of hearing and vocal behavior after hair-cell regeneration
Authors: Dooling R. J. , Ryals B. M. , Manabe K.
Published: July 2002

Postmitotic hair-cell regeneration in the inner ear of birds provides an opportunity to study the effect of renewed auditory input on auditory perception, vocal production, and vocal learning in a vertebrate. We used behavioral conditioning to test both perception and vocal production in a small Australian parrot, the budgerigar. Results show that both auditory perception and vocal production are disrupted when hair cells are damaged or lost but that these behaviors return to near normal over time. Precision in vocal production completely recovers well before recovery of full auditory function. These results may have particular relevance for understanding the relation between hearing loss and human speech production especially where there is consideration of an auditory prosthetic device. The present results show, at least for a bird, that even limited recovery of auditory input soon after deafening can support full recovery of vocal precision.
Role of the
Xlim-1
and
Xbra
genes in anteroposterior patterning of neural tissue by the head and trunk organizer
Authors: Taira Masanori , Saint-Jeannet Jean-Pierre , Dawid Igor B.
Published: July 2002

Anteroposterior patterning of neural tissue is thought to be directed by the axial mesoderm which is functionally divided into head and trunk organizer. The LIM class homeobox gene
Xlim-1
is expressed in the entire axial mesoderm, whereas the distinct transcription factor
Xbra
is expressed in the notochord but not in the prechordal mesoderm. mRNA injection experiments showed that
Xenopus
animal explants (caps) expressing an activated form of Xlim-1 (a LIM domain mutant named 3m) induce anterior neural markers whereas caps coexpressing Xlim-1/3m and Xbra induce posterior neural markers. These data indicate that, in terms of neural inducing ability, Xlim-1/3m-expressing caps correspond to the head organizer and Xlim-1/3m plus Xbra-coexpressing caps to the trunk organizer. Thus the expression domains of
Xlim-1
and
Xbra
correlate with, and possibly define, the functional domains of the organizer. In animal caps Xlim-1/3m initiates expression of a neuralizing factor, chordin, whereas Xbra activates embryonic fibroblast growth factor (eFGF) expression, as reported previously; these factors could mediate the neural inducing and patterning effects that were observed. A dominant-negative FGF receptor (XFD) inhibits posteriorization by Xbra in a dose-dependent manner, supporting the suggestion that eFGF or a related factor has posteriorizing influence.
d
-aspartate localizations imply neuronal and neuroendocrine roles
Authors: Schell Michael J. , Cooper Odelia B. , Snyder Solomon H.
Published: July 2002

Though
l
-amino acids predominate in living organisms, substantial levels of free
d
-serine and
d
-aspartate occur in mammals, especially in nervous and endocrine tissues. Using an antibody specific for glutaraldehyde-fixed
d
-aspartate, we have localized
d
-aspartate in rat tissues. In the brain we observe discrete neuronal localizations of
d
-aspartate, especially in the external plexiform layer of the olfactory bulb, hypothalamic supraoptic and paraventricular nuclei, the medial habenula, and certain brainstem nuclei. In rats 3–4 weeks old, we observe
d
-aspartate in septal nuclei and in a subset of stellate and basket cells of the cerebellum.
d
-aspartate is also concentrated in glands, including the epinephrine cells of the adrenal medulla, the posterior pituitary, and the pineal gland. Levels in the pineal gland are the highest of any mammalian tissue.
d
-aspartate oxidase, visualized by enzyme histochemistry, is concentrated in neurons of the hippocampus, cerebral cortex, and olfactory epithelium, as well as choroid plexus and ependyma. Localizations of
d
-aspartate oxidase are reciprocal to
d
-aspartate, suggesting that the enzyme depletes endogenous stores of the amino acid and might inactivate synaptically released
d
-aspartate.
Establishment of methylation-sensitive–representational difference analysis and isolation of hypo- and hypermethylated genomic fragments in mouse liver tumors
Authors: Ushijima Toshikazu , Morimura Keiichirou , Hosoya Yoko , Okonogi Hideo , Tatematsu Masae , Sugimura Takashi , Nagao Minako
Published: July 2002

Methylation of CpG sites in the genome, which is generally conserved during cell replication, is considered to play important roles in cell differentiation and carcinogenesis. However, investigations on changes in methylation status have been limited to known genes. To make a genome-wide search for differentially methylated genes, we developed a methylation-sensitive–representational difference analysis (MS-RDA) method. The representation of the genome was prepared using the methylation-sensitive restriction enzyme
Hpa
II, and the mixture ratio of tester and driver DNAs was optimized to detect differences in methylation status of a single copy per diploid mammalian genome. By performing comparative MS-RDA of one hepatocellular carcinoma and of background liver tissue of one mouse treated with a food carcinogen (2-amino-3,4-dimethylimidazo[4,5-
f
]quinoline), we were able to identify (
i
) extensive hypomethylation of long interspersed nuclear element repetitive sequences in a number of hepatocellular carcinomas, (
ii
) reduction of the gene dosage of their mitochondrial DNA, and (
iii
) a hypermethylated DNA fragment of unknown origin. Furthermore, by adding the clones obtained in the first MS-RDA to the driver DNA [MS-RDA
w
ith
e
limination of
e
xcessive
c
lones (MS-RDA-WEEC)], nine DNA fragments that could not be detected at the first MS-RDA were isolated as differentially methylated DNA fragments. MS-RDA, combined with MS-RDA-WEEC, is thus a promising approach to identify DNA fragments differentially methylated in two DNA sources.
Two-dimensional confocal images of organization, density, and gating of focal Ca
2+
release sites in rat cardiac myocytes
Authors: Cleemann Lars , Wang Wei , Morad Martin
Published: July 2002

In cardiac myocytes Ca
2+
cross-signaling between Ca
2+
channels and ryanodine receptors takes place by exchange of Ca
2+
signals in microdomains surrounding dyadic junctions, allowing first the activation and then the inactivation of the two Ca
2+
-transporting proteins. To explore the details of Ca
2+
signaling between the two sets of receptors we measured the two-dimensional cellular distribution of Ca
2+
at 240 Hz by using a novel confocal imaging technique. Ca
2+
channel-triggered Ca
2+
transients could be resolved into dynamic “Ca
2+
stripes” composed of hundreds of discrete focal Ca
2+
releases, appearing as bright fluorescence spots (radius ≅ 0.5 μm) at reproducible sites, which often coincided with t-tubules as visualized with fluorescent staining of the cell membrane. Focal Ca
2+
releases triggered stochastically by Ca
2+
current (I
Ca
) changed little in duration (≅7 ms) and size (≅100,000 Ca ions) between −40 and +60 mV, but their frequency of activation and first latency mirrored the kinetics and voltage dependence of I
Ca
. The resolution of 0.95 ± 0.13 reproducible focal Ca
2+
release sites per μm
3
in highly Ca
2+
-buffered cells, where diffusion of Ca
2+
is limited to 50 nm, suggests the presence of about one independent, functional Ca
2+
release site per half sarcomere. The density and distribution of Ca
2+
release sites suggest they correspond to dyadic junctions. The abrupt onset and termination of focal Ca
2+
releases indicate that the cluster of ryanodine receptors in individual dyadic junctions may operate in a coordinated fashion.
Cellular immune response to adenoviral vector infected cells does not requirede novoviral gene expression: Implications for gene therapy
Authors: Kafri Tal , Morgan David , Krahl Troy , Sarvetnick Nora , Sherman Linda , Verma Inder
Published: July 2002

Replication-defective adenoviral (RDAd) vectors can be generated at high titers and infect both dividing and nondividing cells. Long term expression in the transduced tissue, however, has been a problem because of the cellular immune responses against the infected cells. We demonstrate that mice injected with RDAd vectors containing mouse leptin gene reduce food intake and lose weight for only 7 to 10 days. Splenocytes obtained from infected mice are able to lyse target cells infected with RDAd vectors. Surprisingly, target cells infected with psoralen-treated, UV-crosslinked, biologically inactive RDAd also were lysed efficiently by the effector cells. Furthermore, splenocytes obtained from mice injected with inactive RDAd vectors efficiently lysed target cells infected with RDAd vectors. Whether RDAd vectors were injected i.m. or i.v. or through an i.p. route, the extent of lysis was similar. We propose that cells infected with RDAd vectors present antigens for recognition by class 1 major histocompatibility complex-restricted cytotoxic T lymphocytes by a mechanism that does not require viral replication orde novoprotein synthesis. These results should prompt reevaluation of the use of RDAd vectors for gene therapy when long-term expression is required.
Prion protein NMR structure and familial human spongiform encephalopathies
Authors: Riek Roland , Wider Gerhard , Billeter Martin , Hornemann Simone , Glockshuber Rudi , Wüthrich Kurt
Published: July 2002

The refined NMR structure of the mouse prion protein domain
m
PrP(121–231) and the recently reported NMR structure of the complete 208-residue polypeptide chain of
m
PrP are used to investigate the structural basis of inherited human transmissible spongiform encephalopathies. In the cellular form of
m
PrP no spatial clustering of mutation sites is observed that would indicate the existence of disease-specific subdomains. A hydrogen bond between residues 128 and 178 provides a structural basis for the observed highly specific influence of a polymorphism in position 129 in human PrP on the disease phenotype that segregates with the mutation Asp-178–Asn. Overall, the NMR structure implies that only part of the disease-related amino acid replacements lead to reduced stability of the cellular form of PrP, indicating that subtle structural differences in the mutant proteins may affect intermolecular signaling in a variety of different ways.
Fas ligand gene transfer to the vessel wall inhibits neointima formation and overrides the adenovirus-mediated T cell response
Authors: Sata Masataka , Perlman Harris , Muruve Daniel A. , Silver Marcy , Ikebe Mitsuo , Libermann Towia A. , Oettgen Peter , Walsh Kenneth
Published: July 2002

Proliferation of vascular smooth muscle cells (VSMCs) in response to injury plays a key role in the pathogenesis of vascular disorders. Fas ligand (FasL) induces apoptosis in Fas-bearing cells, and its expression on activated T cells contributes to the regulation of the immune response and physiological cell turnover. Here, we show that a replication-defective adenovirus encoding FasL (Ad-FasL) induced apoptosis in Fas-bearing VSMCs. When introduced locally to balloon-injured rat carotid arteries, a well characterized model of a VSMC-derived lesion, Ad-FasL functioned as a potent inhibitor of neointima formation. In rats immunized with an empty adenoviral vector, robust T cell infiltration of the vessel wall was detected after local delivery of a β-galactosidase-expressing virus (Ad-βgal), whereas T cell infiltrates were not detected after local delivery of Ad-FasL. Prior immunization prevented β-galactosidase expression from Ad-βgal, whereas the expression of the FasL transgene was unaffected. When Ad-βgal and Ad-FasL were delivered together to preimmunized animals, T cell infiltration was reduced and β-galactosidase expression was restored. These data demonstrate that Fas ligand gene transfer can effectively inhibit injury-induced vessel lesion formation and can allow adenovirus-harboring cells to evade immune destruction.
Quantitative trait loci and metabolic pathways
Authors: McMullen M. D. , Byrne P. F. , Snook M. E. , Wiseman B. R. , Lee E. A. , Widstrom N. W. , Coe E. H.
Published: July 2002

The interpretation of quantitative trait locus (QTL) studies is
limited by the lack of information on metabolic pathways leading to
most economic traits. Inferences about the roles of the underlying
genes with a pathway or the nature of their interaction with other loci
are generally not possible. An exception is resistance to the corn
earworm
Helicoverpa zea
(Boddie) in maize (
Zea
mays
L.) because of maysin, a C-glycosyl flavone synthesized in
silks via a branch of the well characterized flavonoid pathway. Our
results using flavone synthesis as a model QTL system indicate:
(
i
) the importance of regulatory loci as QTLs,
(
ii
) the importance of interconnecting biochemical
pathways on product levels, (
iii
) evidence for
“channeling” of intermediates, allowing independent synthesis of
related compounds, (
iv
) the utility of QTL analysis in
clarifying the role of specific genes in a biochemical pathway, and
(
v
) identification of a previously unknown locus on
chromosome 9S affecting flavone level. A greater understanding of the
genetic basis of maysin synthesis and associated corn earworm
resistance should lead to improved breeding strategies. More broadly,
the insights gained in relating a defined genetic and biochemical
pathway affecting a quantitative trait should enhance interpretation of
the biological basis of variation for other quantitative traits.
The crystal structure of
Pyrococcus furiosus
ornithine carbamoyltransferase reveals a key role for oligomerization in enzyme stability at extremely high temperatures
Authors: Villeret Vincent , Clantin Bernard , Tricot Catherine , Legrain Christianne , Roovers Martine , Stalon Victor , Glansdorff Nicolas , Van Beeumen Jozef
Published: July 2002

The
Pyrococcus furiosus
(PF) ornithine carbamoyltransferase (OTCase; EC
2.1.3.3
) is an extremely heat-stable enzyme that maintains about 50% of its activity after heat treatment for 60 min at 100°C. To understand the molecular basis of thermostability of this enzyme, we have determined its three-dimensional structure at a resolution of 2.7 Å and compared it with the previously reported structures of OTCases isolated from mesophilic bacteria. Most OTCases investigated up to now are homotrimeric and devoid of allosteric properties. A striking exception is the catabolic OTCase from
Pseudomonas aeruginosa
, which is allosterically regulated and built up of four trimers disposed in a tetrahedral manner, an architecture that actually underlies the allostery of the enzyme. We now report that the thermostable PF OTCase (420 kDa) presents the same 23-point group symmetry. The enzyme displays Michaelis–Menten kinetics. A detailed comparison of the two enzymes suggests that, in OTCases, not only allostery but also thermophily was achieved through oligomerization of a trimer as a common catalytic motif. Thermal stabilization of the PF OTCase dodecamer is mainly the result of hydrophobic interfaces between trimers, at positions where allosteric binding sites have been identified in the allosteric enzyme. The present crystallographic analysis of PF OTCase provides a structural illustration that oligomerization can play a major role in extreme thermal stabilization.
All-or-none potentiation at CA3-CA1 synapses
Authors: Petersen Carl C. H. , Malenka Robert C. , Nicoll Roger A. , Hopfield John J.
Published: July 2002

The molecular mechanisms underlying long-term potentiation in the hippocampus have received much attention because of the likely functional importance of synaptic plasticity for information storage and the development of neuronal connectivity. Surprisingly, it remains unclear whether activity modifies the strength of individual synapses in a digital (all-or-none) or analog (graded) manner. Here we characterize step-like all-or-none transitions from baseline synaptic transmission to potentiated states following protocols for inducing potentiation at putative single CA3-CA1 synaptic connections. Individual synapses appear to have all-or-none potentiation indicative of highly cooperative processes but different thresholds for undergoing potentiation. These results raise the possibility that some forms of synaptic memory may be stored in a digital manner in the brain.
CD40 ligand (CD154) stimulation of macrophages to produce HIV-1-suppressive β-chemokines
Authors: Kornbluth Richard S. , Kee Kristin , Richman Douglas D.
Published: July 2002

β-chemokines play an important role in the development of immunologic reactions. Macrophages are major β-chemokine-producing cells during T-cell directed, delayed-type hypersensitivity reactions in tissues, and have been reported to be important producers of β-chemokines in the lymph nodes of HIV-1-infected individuals. However, the physiological signals responsible for inducing macrophages to produce β-chemokines have not been established. Two soluble T cell products, interferon-γ and granulocyte-macrophage colony stimulating factor, were added to cultured macrophages, but failed to stimulate the production of macrophage inflammatory protein-1α and -1β; regulated upon activation, normal T cell expressed and secreted (RANTES); or monocyte chemoattractant protein-1. Instead, direct cell–cell contact between macrophages and cells engineered to express CD40L (also known as CD154) resulted in the production of large amounts of macrophage inflammatory protein-1α and -1β, and RANTES (all ligands for CCR5), and monocyte chemoattractant protein-1 (a ligand for CCR2). Supernatants from CD40L-stimulated macrophages protected CD4+T cells from infection by a nonsyncytium-inducing strain of HIV-1 (which uses CCR5 as a coreceptor). These results have implications for granulomatous diseases, and conditions such as atherosclerosis and multiple sclerosis, where CD40L-bearing cells have been found in the macrophage-rich lesions where β-chemokines are being produced. Overall, these findings define a pathway linking the specific recognition of antigen by T cells to the production of β-chemokines by macrophages. This pathway may play a role in anti-HIV-1 immunity and the development of immunologic reactions or lesions.
The telomerase RNA pseudoknot is critical for the stable assembly of a catalytically active ribonucleoprotein
Authors: Gilley David , Blackburn Elizabeth H.
Published: July 2002

Telomerase is a ribonucleoprotein reverse transcriptase that synthesizes telomeric DNA. A pseudoknot structure is phylogenetically conserved within the RNA component of telomerase in all ciliated protozoans examined. Here, we report that disruptions of the pseudoknot base pairing within the telomerase RNA from
Tetrahymena thermophila
prevent the stable assembly
in vivo
of an active telomerase. Restoring the base-pairing potential of the pseudoknot by compensatory changes restores telomerase activity to essentially wild-type levels. Therefore, the pseudoknot topology rather than sequence is critical for an active telomerase. Furthermore, we show that disruption of the pseudoknot prevents the association of the RNA with the reverse transcriptase protein subunit of telomerase. Thus, we provide an example of a structural motif within the telomerase RNA that is required for telomerase function and identify the domain that is required for telomerase complex formation. Hence, we identify a biological role for a pseudoknot: promoting the stable assembly of a catalytically active ribonucleoprotein.
Hemoglobin induction in mouse macrophages
Authors: Liu Limin , Zeng Ming , Stamler Jonathan S.
Published: July 2002

The common perception that hemoglobin is involved solely in the transport of oxygen and carbon dioxide has been challenged by recent studies with nitric oxide (NO). These studies have shown that the primordial bacterial flavohemoglobin functions to consume NO enzymatically (to protect from nitrosative stress), whereas mammalian hemoglobin functions to deliver NO (thus maximizing oxygen delivery in the respiratory cycle). Here we report that murine macrophages stimulated to produce NO with lipopolysaccharide and interferon-γ express the βminorhemoglobin subunit. Consumption of NO, however, was not increased by cytokines or by hemoglobin expression. These data suggest alternative functions for globins in mammalian cells, and they challenge the prevailing view that the expression of α- and β-globin genes is always balanced and coordinated.
Recombinant Semliki Forest virus and Sindbis virus efficiently infect neurons in hippocampal slice cultures
Authors: Ehrengruber Markus U. , Lundstrom Kenneth , Schweitzer Christophe , Heuss Christian , Schlesinger Sondra , Gähwiler Beat H.
Published: July 2002

Gene transfer into nervous tissue is a powerful tool for the analysis of gene function. By using a rat hippocampal slice culture preparation, we show here that Semliki Forest virus (SFV) and Sindbis virus (SIN) vectors are useful for the effective infection of neurons. The stratum pyramidale and/or the granular cell layer were injected with recombinant virus encoding β-galactosidase (LacZ) or green fluorescent protein (GFP). By using low concentrations of injected SFV-LacZ or SIN-LacZ, we detected LacZ staining of pyramidal cells, interneurons, and granule cells. About 60% of the infected cells showed clear neuronal morphology; thus, relatively few glial cells expressed the transgene. Expression of GFP from SFV and SIN vectors gave similar results, with an even higher percentage (>90%) of the GFP-positive cells identified as neurons. Infected pyramidal cells were readily recognized in living slices, displaying GFP fluorescence in dendrites of up to fourth order and in dendritic spines. They appeared morphologically normal and viable at 1–5 days postinfection. We conclude that both SFV and SIN vectors efficiently transfer genes into neurons in hippocampal slice cultures. In combination with the GFP reporter, SFV and SIN vectors will allow the physiological examination of identified neurons that have been modified by overexpression or suppression of a specific gene product.
Molecular characterization of H9N2 influenza viruses: Were they the donors of the “internal” genes of H5N1 viruses in Hong Kong?
Authors: Guan Yi , Shortridge Kennedy F. , Krauss Scott , Webster Robert G.
Published: July 2002

The origin of the H5N1 influenza viruses that killed six of eighteen infected humans in 1997 and were highly pathogenic in chickens has not been resolved. These H5N1 viruses transmitted directly to humans from infected poultry. In the poultry markets in Hong Kong, both H5N1 and H9N2 influenza viruses were cocirculating, raising the possibility of genetic reassortment. Here we analyze the antigenic and genetic features of H9N2 influenza viruses with different epidemiological backgrounds. The results suggest that the H9N2 influenza viruses of domestic ducks have become established in the domestic poultry of Asia. Phylogenetic and antigenic analyses of the H9N2 viruses isolated from Hong Kong markets suggest three distinct sublineages. Among the chicken H9N2 viruses, six of the gene segments were apparently derived from an earlier chicken H9N2 virus isolated in China, whereas the PB1 and PB2 genes are closely related to those of the H5N1 viruses and a quail H9N2 virus—A/quail/Hong Kong/G1/97 (Qa/HK/G1/97)—suggesting that many of the 1997 chicken H9 isolates in the markets were reassortants. The similarity of the internal genes of Qa/HK/G1/97 virus to those of the H5N1 influenza viruses suggests that the quail virus may have been the internal gene donor. Our findings indicate that the human and poultry H5N1 influenza viruses in Hong Kong in 1997 were reassortants that obtained internal gene segments from Qa/HK/G1/97. However, we cannot be certain whether the replicate complex of H5N1 originated from Qa/HK/G1/97 or whether the reverse transfer occurred; the available evidence supports the former proposal.
Human Dmc1 protein binds DNA as an octameric ring
Authors: Passy Sophia I. , Yu Xiong , Li Zhufang , Radding Charles M. , Masson Jean-Yves , West Stephen C. , Egelman Edward H.
Published: July 2002

The bacterial RecA protein has been the most intensively studied enzyme in homologous genetic recombination. The core of RecA is structurally homologous to that of the F1-ATPase and helicases. Like the F1-ATPase and ring helicases, RecA forms a hexameric ring. The human Dmc1 (hDmc1) protein, a meiosis-specific recombinase, is homologous to RecA. We show that hDmc1 forms octameric rings. Unlike RecA and Rad51, however, hDmc1 protein does not form helical filaments. The hDmc1 ring binds DNA in the central channel, as do the ring helicases, which is likely to represent the active form of the protein. These observations indicate that the conservation of the RecA-like ring structure extends from bacteria to humans, and that some RecA homologs may form both rings and filaments, whereas others may function only as rings.
Human artificial chromosomes generated by modification of a yeast artificial chromosome containing both human alpha satellite and single-copy DNA sequences
Authors: Henning Karla A. , Novotny Elizabeth A. , Compton Sheila T. , Guan Xin-Yuan , Liu Pu P. , Ashlock Melissa A.
Published: July 2002

A human artificial chromosome (HAC) vector was constructed from a 1-Mb yeast artificial chromosome (YAC) that was selected based on its size from among several YACs identified by screening a randomly chosen subset of the Centre d’Étude du Polymorphisme Humain (CEPH) (Paris) YAC library with a degenerate alpha satellite probe. This YAC, which also included non-alpha satellite DNA, was modified to contain human telomeric DNA and a putative origin of replication from the human β-globin locus. The resultant HAC vector was introduced into human cells by lipid-mediated DNA transfection, and HACs were identified that bound the active kinetochore protein CENP-E and were mitotically stable in the absence of selection for at least 100 generations. Microdissected HACs used as fluorescence
in situ
hybridization probes localized to the HAC itself and not to the arms of any endogenous human chromosomes, suggesting that the HAC was not formed by telomere fragmentation. Our ability to manipulate the HAC vector by recombinant genetic methods should allow us to further define the elements necessary for mammalian chromosome function.
XK469, a selective topoisomerase IIβ poison
Authors: Gao Hanlin , Huang Kuan-Chun , Yamasaki Edith F. , Chan Kenneth K. , Chohan Lubna , Snapka Robert M.
Published: July 2002

XK469 (NSC 697887) is a synthetic quinoxaline phenoxypropionic acid
derivative that possesses unusual solid tumor selectivity and activity
against multidrug-resistant cancer cells. We report here that XK469 and
its S(−) and R(+)-isomers induce reversible protein-DNA crosslinks in
mammalian cells. Under protein denaturing conditions, the protein-DNA
crosslinks are rendered irreversible and stable to DNA banding by CsCl
gradient ultracentrifugation. Several lines of evidence indicate that
the primary target of XK469 is topoisomerase IIβ. Preferential
targeting of topoisomerase IIβ may explain the solid tumor
selectivity of XK469 and its analogs because solid tumors, unlike
leukemias, often have large populations of cells in the
G
1
/G
0
phases of the cell cycle in which
topoisomerase IIβ is high whereas topoisomerase IIα, the primary
target of many leukemia selective drugs, is low.
The molecular chaperone Hsp90 can negatively regulate the activity of a glucocorticosteroid-dependent promoter
Authors: Kang Kwang Il , Meng Xia , Devin-Leclerc Jocelyne , Bouhouche Ilham , Chadli Ahmed , Cadepond Françoise , Baulieu Etienne-Emile , Catelli Maria-Grazia
Published: July 2002

Hsp90, a molecular chaperone required for the functioning of
glucocorticosteroid receptor (GR), ensures, by direct interaction, the
conformational competence of the steroid-binding pocket. In addition to
having this positive function, Hsp90 maintains steroid receptors in an
inactive form in the absence of hormone. However, neither the
participation of Hsp90 once the pathway has been activated by the
ligand nor the importance of increased Hsp90 levels in determining the
amplitude of the response has ever been assessed directly. Here, by
increasing the Hsp90/GR ratio in the nuclear compartment, we
found an attenuation of the response to glucocorticosteroids that was
not due to a nonspecific or toxic effect of the Hsp90 modified by
nuclear targeting. Since this negative effect was more pronounced at
high levels of hormone, when receptor and Hsp90 are maximally
dissociated, the possibility of an interaction between Hsp90 and GR,
already activated to a DNA-binding form, was directly investigated.
Indeed GR, after
in vivo
activation by ligand, was still
able to reassociate with Hsp90, suggesting that this interaction plays
a role
in vivo
, possibly in receptor recycling.
Moreover, the GR binding to its DNA response element was inhibited by
an excess of Hsp90, pointing to a function of Hsp90 in the nuclear
compartment. It is thus proposed that an increased Hsp90/GR
ratio influences the responsiveness to ligand at a step that is after
receptor activation. This increased ratio may be of pathophysiological
relevance in the different circumstances that lead to an elevated level
of nuclear Hsp90.
Proteasome-dependent degradation of the human estrogen receptor
Authors: Nawaz Zafar , Lonard David M. , Dennis Andrew P. , Smith Carolyn L. , O’Malley Bert W.
Published: July 2002

In eukaryotic cells, the ubiquitin–proteasome pathway is the major mechanism for the targeted degradation of proteins with short half-lives. The covalent attachment of ubiquitin to lysine residues of targeted proteins is a signal for the recognition and rapid degradation by the proteasome, a large multi-subunit protease. In this report, we demonstrate that the human estrogen receptor (ER) protein is rapidly degraded in mammalian cells in an estradiol-dependent manner. The treatment of mammalian cells with the proteasome inhibitor MG132 inhibits activity of the proteasome and blocks ER degradation, suggesting that ER protein is turned over through the ubiquitin–proteasome pathway. In addition, we show that
in vitro
ER degradation depends on ubiquitin-activating E1 enzyme (UBA) and ubiquitin-conjugating E2 enzymes (UBCs), and the proteasome inhibitors MG132 and lactacystin block ER protein degradation
in vitro
. Furthermore, the UBA/UBCs and proteasome inhibitors promote the accumulation of higher molecular weight forms of ER. The UBA and UBCs, which promote ER degradation
in vitro
, have no significant effect on human progesterone receptor and human thyroid hormone receptor β proteins.
Innate and acquired humoral immunities to influenza virus are mediated by distinct arms of the immune system
Authors: Baumgarth Nicole , Herman Ometa C. , Jager Gina C. , Brown Lorena , Herzenberg Leonard A. , Herzenberg Leonore A.
Published: July 2002

“Natural” Igs, mainly IgM, comprise part of the innate immune system present in healthy individuals, including antigen-free mice. These Igs are thought to delay pathogenicity of infecting agents until antigen-induced high affinity Igs of all isotypes are produced. Previous studies suggested that the acquired humoral response arises directly from the innate response, i.e., that B cells expressing natural IgM, upon antigen encounter, differentiate to give rise both to cells that secrete high amounts of IgM and to cells that undergo affinity maturation and isotype switching. However, by using a murine model of influenza virus infection, we demonstrate here that the B cells that produce natural antiviral IgM neither increase their IgM production nor undergo isotype switching to IgG2a in response to the infection. These cells are distinct from the B cells that produce the antiviral response after encounter with the pathogen. Our data therefore demonstrate that the innate and the acquired humoral immunities to influenza virus are separate effector arms of the immune system and that antigen exposureper seis not sufficient to increase natural antibody production.
Transdermal photopolymerization for minimally invasive implantation
Authors: Elisseeff J. , Anseth K. , Sims D. , McIntosh W. , Randolph M. , Langer R.
Published: July 2002

Photopolymerizations are widely used in medicine to create polymer networks for use in applications such as bone restorations and coatings for artificial implants. These photopolymerizations occur by directly exposing materials to light in “open” environments such as the oral cavity or during invasive procedures such as surgery. We hypothesized that light, which penetrates tissue including skin, could cause a photopolymerization indirectly. Liquid materials then could be injected s.c. and solidified by exposing the exterior surface of the skin to light. To test this hypothesis, the penetration of UVA and visible light through skin was studied. Modeling predicted the feasibility of transdermal polymerization with only 2 min of light exposure required to photopolymerize an implant underneath human skin. To establish the validity of these modeling studies, transdermal photopolymerization first was applied to tissue engineering by using “injectable” cartilage as a model system. Polymer/chondrocyte constructs were injected s.c. and transdermally photopolymerized. Implants harvested at 2, 4, and 7 weeks demonstrated collagen and proteoglycan production and histology with tissue structure comparable to native neocartilage. To further examine this phenomenon and test the applicability of transdermal photopolymerization for drug release devices, albumin, a model protein, was released for 1 week from photopolymerized hydrogels. With further study, transdermal photpolymerization potentially could be used to create a variety of new, minimally invasive surgical procedures in applications ranging from plastic and orthopedic surgery to tissue engineering and drug delivery.