bluuebunny/crossref_metadata_2025 huggingface dataset:
Preferential synaptic relationships between substance P-immunoreactive boutons and neurokinin 1 receptor sites in the rat spinal cord
Authors: McLeod A. L. , Krause J. E. , Cuello A. C. , Ribeiro-da-Silva A.
Published: July 2002

Substance P plays an important role in the transmission of pain-related information in the dorsal horn of the spinal cord. Recent immunocytochemical studies have shown a mismatch between the distribution of substance P and its receptor in the superficial laminae of the dorsal horn. Because such a mismatch was not observed by using classical radioligand binding studies, we decided to investigate further the issue of the relationship between substance P and its receptor by using an antibody raised against a portion of the carboxyl terminal of the neurokinin 1 receptor and a bispecific monoclonal antibodies against substance P and horseradish peroxidase. Light microscopy revealed a good correlation between the distributions of substance P and the neurokinin 1 receptor, both being localized with highest densities in lamina I and outer lamina II of the spinal dorsal horn. An ultrastructural double-labeling study, combining preembedding immunogold with enzyme-based immunocytochemistry, showed that most neurokinin 1 receptor immunoreactive dendrites were apposed by substance P containing boutons. A detailed quantitative analysis revealed that neurokinin 1 receptor immunoreactive dendrites received more appositions and synapses from substance P immunoreactive terminals than those not expressing the neurokinin 1 receptor. Such preferential innervation by substance P occurred in all superficial dorsal horn laminae even though neurokinin 1 receptor immunoreactive dendrites were a minority of the total number of dendritic profiles in the above laminae. These results suggest that, contrary to the belief that neuropeptides act in a diffuse manner at a considerable distance from their sites of release, substance P should act on profiles expressing the neurokinin 1 receptor at a short distance from its site of release.
Intranuclear targeting of AML/CBFα regulatory factors to nuclear matrix-associated transcriptional domains
Authors: Zeng Congmei , McNeil Sandra , Pockwinse Shirwin , Nickerson Jeffrey , Shopland Lindsay , Lawrence Jeanne B. , Penman Sheldon , Hiebert Scott , Lian Jane B. , van Wijnen André J. , Stein Janet L. , Stein Gary S.
Published: July 2002

The AML/CBFα
runt
transcription factors are key regulators of hematopoietic and bone tissue-specific gene expression. These factors contain a 31-amino acid nuclear matrix targeting signal that supports association with the nuclear matrix. We determined that the AML/CBFα factors must bind to the nuclear matrix to exert control of transcription. Fusing the nuclear matrix targeting signal to the GAL4 DNA binding domain transactivates a genomically integrated GAL4 responsive reporter gene. These data suggest that AML/CBFα must associate with the nuclear matrix to effect transcription. We used fluorescence labeling of epitope-tagged AML-1B (CBFA2) to show it colocalizes with a subset of hyperphosphorylated RNA polymerase II molecules concentrated in foci and linked to the nuclear matrix. This association of AML-1B with RNA polymerase II requires active transcription and a functional DNA binding domain. The nuclear matrix domains that contain AML-1B are distinct from SC35 RNA processing domains. Our results suggest two of the requirements for AML-dependent transcription initiation by RNA polymerase II are association of AML-1B with the nuclear matrix together with specific binding of AML to gene promoters.
Agrobacterium-transformed rice plants expressing syntheticcryIA(b)andcryIA(c)genes are highly toxic to striped stem borer and yellow stem borer
Authors: Cheng Xiongying , Sardana Ravinder , Kaplan Harvey , Altosaar Illimar
Published: July 2002

Over 2,600 transgenic rice plants in nine strains were regenerated from >500 independently selected hygromycin-resistant calli afterAgrobacterium-mediated transformation. The plants were transformed with fully modified (plant codon optimized) versions of two syntheticcryIA(b)andcryIA(c)coding sequences fromBacillus thuringiensisas well as thehphandgusgenes, coding for hygromycin phosphotransferase and β-glucuronidase, respectively. These sequences were placed under control of the maizeubiquitinpromoter, the CaMV35S promoter, and theBrassica Bp10gene promoter to achieve high and tissue-specific expression of the lepidopteran-specific δ-endotoxins. The integration, expression, and inheritance of these genes were demonstrated in R0and R1generations by Southern, Northern, and Western analyses and by other techniques. Accumulation of high levels (up to 3% of soluble proteins) of CryIA(b) and CryIA(c) proteins was detected in R0plants. Bioassays with R1transgenic plants indicated that the transgenic plants were highly toxic to two major rice insect pests, striped stem borer (Chilo suppressalis) and yellow stem borer (Scirpophaga incertulas), with mortalities of 97–100% within 5 days after infestation, thus offering a potential for effective insect resistance in transgenic rice plants.
Architectural specificity in chromatin structure at the TATA box
in vivo
: Nucleosome displacement upon β-phaseolin gene activation
Authors: Li Guofu , Chandler Simon P. , Wolffe Alan P. , Hall Timothy C.
Published: July 2002

Extensive studies of the β-phaseolin (
phas
) gene in transgenic tobacco have shown that it is highly active during seed embryogenesis but is completely silent in leaf and other vegetative tissues.
In vivo
footprinting revealed that the lack of even basal transcriptional activity in vegetative tissues is associated with the presence of a nucleosome that is rotationally positioned with base pair precision over three phased TATA boxes present in the
phas
promoter. Positioning is sequence-dependent because an identical rotational setting is obtained upon nucleosome reconstitution
in vitro
. A comparison of DNase I and dimethyl sulfate footprints
in vivo
and
in vitro
strongly suggests that this repressive chromatin architecture is remodeled concomitant with gene activation in the developing seed. This leads to the disruption of histone-mediated DNA wrapping and the assembly of the TATA boxes into a transcriptionally competent nucleoprotein complex.
Endogenous cellulases in animals: Isolation of β-1,4-endoglucanase genes from two species of plant-parasitic cyst nematodes
Authors: Smant Geert , Stokkermans Jack P. W. G. , Yan Yitang , de Boer Jan M. , Baum Thomas J. , Wang Xiaohong , Hussey Richard S. , Gommers Fred J. , Henrissat Bernard , Davis Eric L. , Helder Johannes , Schots Arjen , Bakker Jaap
Published: July 2002

β-1,4-Endoglucanases (EGases, EC
3.2.1.4
) degrade polysaccharides possessing β-1,4-glucan backbones such as cellulose and xyloglucan and have been found among extremely variegated taxonomic groups. Although many animal species depend on cellulose as their main energy source, most omnivores and herbivores are unable to produce EGases endogenously. So far, all previously identified EGase genes involved in the digestive system of animals originate from symbiotic microorganisms. Here we report on the synthesis of EGases in the esophageal glands of the cyst nematodes
Globodera rostochiensis
and
Heterodera glycines
. From each of the nematode species, two cDNAs were characterized and hydrophobic cluster analysis revealed that the four catalytic domains belong to family 5 of the glycosyl hydrolases (EC 3.2.1, 3.2.2, and 3.2.3). These domains show 37–44% overall amino acid identity with EGases from the bacteria
Erwinia chrysanthemi
,
Clostridium acetobutylicum
, and
Bacillus subtilis
. One EGase with a bacterial type of cellulose-binding domain was identified for each nematode species. The leucine-rich hydrophobic core of the signal peptide and the presence of a polyadenylated 3′ end precluded the EGases from being of bacterial origin. Cyst nematodes are obligatory plant parasites and the identified EGases presumably facilitate the intracellular migration through plant roots by partial cell wall degradation.
Iron-dependent oxidation, ubiquitination, and degradation of iron regulatory protein 2: Implications for degradation of oxidized proteins
Authors: Iwai Kazuhiro , Drake Steven K. , Wehr Nancy B. , Weissman Allan M. , LaVaute Timothy , Minato Nagahiro , Klausner Richard D. , Levine Rodney L. , Rouault Tracey A.
Published: July 2002

The ability of iron to catalyze formation of reactive oxygen species significantly contributes to its toxicity in cells and animals. Iron uptake and distribution is regulated tightly in mammalian cells, in part by iron regulatory protein 2 (IRP2), a protein that is degraded efficiently by the proteasome in iron-replete cells. Here, we demonstrate that IRP2 is oxidized and ubiquitinated in cells before degradation. Moreover, iron-dependent oxidation converts IRP2 into a substrate for ubiquitination
in vitro
. A regulatory pathway is described in which excess iron is sensed by its ability to catalyze site-specific oxidations in IRP2, oxidized IRP2 is ubiquitinated, and ubiquitinated IRP2 subsequently is degraded by the proteasome. Selective targeting and removal of oxidatively modified proteins may contribute to the turnover of many proteins that are degraded by the proteasome.
Transposition of the
mariner
element from
Drosophila mauritiana
in zebrafish
Authors: Fadool James M. , Hartl Daniel L. , Dowling John E.
Published: July 2002

With the increased popularity of zebrafish (
Danio rerio
) for mutagenesis studies, efficient methods for manipulation of its genome are needed. One approach is the use of a transposable element as a vector for gene transfer in this species. We report here the transformation of zebrafish and germ-line transmission of the
mariner
element from
Drosophila mauritiana
. The
mariner
element was selected because its transposition is independent of host-specific factors. One- to two-cell-stage zebrafish embryos were coinjected with a supercoiled plasmid carrying the nonautonomous
mariner
element
peach
and mRNA encoding the transposase. Surviving larvae were reared to adulthood, and the transmission of
peach
to the F
1
generation was tested by PCR. Four of the 12 founders, following plasmid injections on 2 different days, transmitted the element to their progeny. Inheritance of the transgene from the F
1
to the F
2
generation showed a Mendelian pattern. No plasmid sequences were detected by PCR or Southern blot analysis, indicating transposition of
peach
rather than random integration of the plasmid DNA. These data provide evidence of transformation of a vertebrate with a transposable element and support the host-independent mechanism for transposition of the
mariner
element. We suggest this system could be used for insertional mutagenesis or for identifying active regions of the genome in the zebrafish.
Identification of a meiosis-specific protein as a member of the class of cancer/testis antigens
Authors: Türeci Özlem , Sahin Ugur , Zwick Carsten , Koslowski Michael , Seitz Gerhard , Pfreundschuh Michael
Published: July 2002

Little is known about the function of human cancer/testis antigens (CTAs), such as MAGE, BAGE, GAGE, HOM-MEL-40, and NY-ESO-1, the expression of which is restricted to human malignancies and testis. When screening a cDNA expression library enriched for testis-specific representative long transcripts for reactivity with high-titered IgG antibodies from the serum of a patient with renal cell carcinoma, one repeatedly detected antigen, designated HOM-TES-14, turned out to be encoded by the synaptonemal complex protein 1 (SCP-1) gene. SCP-1 is known to be selectively expressed during the meiotic prophase of spermatocytes and is involved in the pairing of homologous chromosomes, an essential step for the generation of haploid cells in meiosis I. Investigation of a broad spectrum of normal and malignant tissues revealed expression of SCP-1 transcripts and antigen selectively in a variety of neoplastic tissues and tumor cell lines. Immunofluorescence microscopy analysis with specific antiserum showed a cell cycle phase-independent nuclear expression of SCP-1 protein in cancer cells. SCP-1 differs from other members of the class of CTA by its localization on chromosome 1 and its frequent expression in malignant gliomas, breast, renal cell, and ovarian cancer. The aberrant expression of SCP-1 in tumors might contribute to their genomic instability and suggests that the functional role of other CTA might also relate to meiosis.
Mechanisms by which IκB proteins control NF-κB activity
Authors: Simeonidis Simos , Stauber Deborah , Chen Guoying , Hendrickson Wayne A. , Thanos Dimitris
Published: July 2002

The biological activity of the transcription factor NF-κB is differentially controlled by three IκB proteins, IκBα, IκBβ, and IκBɛ. We have examined the molecular basis for the differential inhibitory strengths of IκB proteins by constructing hybrid IκBs and found that the first ankyrin repeat of IκBα is responsible for its strong inhibitory effect. Swapping a putative β-turn within the first ankyrin repeat between the strong IκBα and the weak IκBβ inhibitors switches their
in vivo
inhibitory activity on NF-κB. The qualitatively distinct contacts made by this β-turn in IκBα and IκBβ with NF-κB determine the efficiency by which IκBs sequester NF-κB to the cytoplasm, thus explaining their distinct effects on gene activity.
Exon repetition in mRNA
Authors: Frantz Simon A. , Thiara Amrik S. , Lodwick David , Ng Leong L. , Eperon Ian C. , Samani Nilesh J.
Published: July 2002

The production of different transcripts (transcript heterogeneity) is a feature of many genes that may result in phenotypic variation. Several mechanisms, that occur at both the DNA and RNA level have been shown to contribute to this transcript heterogeneity in mammals, all of which involve either the rearrangement of sequences within a genome or the use of alternative signals in linear, contiguous DNA or RNA. Here we describe tissue-specific repetition of selective exons in transcripts of a rat gene (SA) with a normal exon–intron organization. We conclude that nonlinear mRNA processing can generate tissue-specific transcripts.
Mutations conferring resistance to phenamil and amiloride, inhibitors of sodium-driven motility of
Vibrio parahaemolyticus
Authors: Jaques Sandford , Kim Yun-Kyeong , McCarter Linda L.
Published: July 2002

The bacterial flagellum is powered by a rotary motor capable of turning the helical flagellar propeller at very high speeds. Energy to drive rotation is derived from the transmembrane electrochemical potential of specific ions. Ions passing through a channel component are thought to generate the force to power rotation. Two kinds of motors, dependent on different coupling ions, have been described: proton-driven and sodium-driven motors. There are four known genes encoding components of the sodium-powered polar flagellar motor in
Vibrio parahaemolyticus
. Two, which are characterized here, are homologous to genes encoding constituents of the proton-type motor (
motA
and
motB
), and two encode components unique to the sodium-type motor (
motX
and
motY
). The sodium-channel-blocking drugs phenamil and amiloride inhibit rotation of the polar flagellum and therefore can be used to probe the architecture of the motor. Mutants were isolated that could swim in the presence of phenamil or amiloride. The majority of the mutations conferring phenamil-resistant motility alter nucleotides in the
motA
or
motB
genes. The resultant amino acid changes localize to the cytoplasmic face of the torque generator and permit identification of potential sodium-interaction sites. Mutations that confer motility in the presence of amiloride do not alter any known component of the sodium-type flagellar motor. Thus, evidence supports the existence of more than one class of sodium-interaction site at which inhibitors can interfere with sodium-driven motility.
Calcineurin and vacuolar-type H+-ATPase modulate macrophage effector functions
Authors: Conboy Irina M. , Manoli Devanand , Mhaiskar Vidula , Jones Patricia P.
Published: July 2002

While effector molecules produced by activated macrophages (including nitric oxide, tumor necrosis factor α, interleukin 1, etc.) help to eliminate pathogens, high levels of these molecules can be deleterious to the host itself. Despite their importance, the mechanisms modulating macrophage effector functions are poorly understood. This work introduces two key negative regulators that control the levels and duration of macrophage cytokine production. Vacuolar-type H+-ATPase (V-ATPase) and calcineurin (Cn) constitutively act in normal macrophages to suppress expression of inflammatory cytokines in the absence of specific activation and to inhibit macrophage cytokine responses induced by bacterial lipopolysaccharide (V-ATPase), interferon γ (V-ATPase and Cn), and calcium (Ca2+) flux (Cn). Cn and V-ATPase modulate effector gene expression at the mRNA level by inhibiting transcription factor NF-κB. This negative regulation by Cn is opposite to its crucial positive role in T cells, where it activates NFAT transcription factor(s) leading to expression of interleukin 2, tumor necrosis factor α, and other cytokine genes. The negative effects of V-ATPase and Cn on NF-κB-dependent gene expression are not limited to the macrophage lineage, as similar effects have been seen with a murine fibroblast cell line and with primary astrocytes.
Widespread intra-serotype recombination in natural populations of dengue virus
Authors: Worobey Michael , Rambaut Andrew , Holmes Edward C.
Published: July 2002

Diversity analysis of 71 published dengue virus gene sequences revealed several strains that appeared to be mosaics comprising gene regions with conflicting evolutionary histories. Subsequent maximum likelihood breakpoint estimation identified seven recombinants, including members of three of the four dengue virus serotypes, with breakpoints in the premembrane/membrane gene, the envelope gene, and at the junction of the envelope and first nonstructural genes. Many of the individual recombinants contain sequence representing separate genetic subtypes. The results were highly statistically significant and were confirmed by phylogenetic analysis of the regions of interest. These findings indicate that recombination may play a very significant role in shaping genetic diversity in dengue virus and, as such, have important implications for its biology and its control.
“Mutagenesis” by peptide aptamers identifies genetic network members and pathway connections
Authors: Geyer C. Ronald , Colman-Lerner Alejandro , Brent Roger
Published: July 2002

We selected peptide aptamers from combinatorial libraries that disrupted cell-cycle arrest caused by mating pheromone in yeast. We used these aptamers as baits in two-hybrid hunts to identify genes involved in cell-cycle arrest. These experiments identified genes known to function in the pathway, as well as a protein kinase, the CBK1 product, whose function was not known. We used a modified two-hybrid system to identify specific interactions disrupted by these aptamers. These experiments demonstrate a means to perform “genetics” on the protein complement of a cell without altering its genetic material. Peptide aptamers can be identified that disrupt a process. These aptamers can then be used as affinity reagents to identify individual proteins and protein interactions needed for the process. Forward genetic analysis with peptide aptamer “mutagens” should be particularly useful in elucidating genetic networks in organisms and processes for which classical genetics is not feasible.
Cytotoxic T lymphocyte antigen-4 (CTLA-4) regulates primary and secondary peptide-specific CD4+T cell responses
Authors: Chambers Cynthia A. , Kuhns Michael S. , Allison James P.
Published: July 2002

CTLA-4-deficient mice develop a fatal lymphoproliferative disorder, characterized by polyclonal expansion of peripheral lymphocytes. To examine the effect of restricting the CD4+TCR repertoire on the phenotype of CTLA-4-deficient mice and to assess the influence of CTLA-4 on peptide-specific CD4+T cell responsesin vitro, an MHC class II-restricted T cell receptor (AND TCR) transgene was introduced into the CTLA-4−/−animals. The expression of the AND TCR transgene by CD4+T cells delays but does not prevent the lymphoproliferation in the CTLA-4−/−mice. The CD4+T cells become preferentially activated and expand. Interestingly, young AND TCR+CTLA-4−/−mice carrying a null mutation in therag-1gene remain healthy and the T cells maintain a naive phenotype until later in life. We demonstrate that CTLA-4 regulates the peptide-specific proliferative response generated by naive and previously activated AND TCR+RAG−/−T cellsin vitro. The absence of CTLA-4 also augments the responder frequency of cytokine-secreting AND TCR+RAG−/−T cells. These results demonstrate that CTLA-4 is a key regulator of peptide-specific CD4+T cell responses and support the model that CTLA-4 plays a differential role in maintaining T cell homeostasis of CD4+vs. CD8+T cells.
Transgenic mice expressing a Huntington’s disease mutation are resistant to quinolinic acid-induced striatal excitotoxicity
Authors: Hansson Oskar , Petersén Åsa , Leist Marcel , Nicotera Pierluigi , Castilho Roger F. , Brundin Patrik
Published: July 2002

Huntington’s disease (HD) is a hereditary neurodegenerative disorder presenting with chorea, dementia, and extensive striatal neuronal death. The mechanism through which the widely expressed mutant HD gene mediates a slowly progressing striatal neurotoxicity is unknown. Glutamate receptor-mediated excitotoxicity has been hypothesized to contribute to the pathogenesis of HD. Here we show that transgenic HD mice expressing exon 1 of a human HD gene with an expanded number of CAG repeats (line R6/1) are strongly protected from acute striatal excitotoxic lesions. Intrastriatal infusions of the
N
-methyl-
d
-aspartate (NMDA) receptor agonist quinolinic acid caused massive striatal neuronal death in wild-type mice, but no damage in transgenic HD littermates. The remarkable neuroprotection in transgenic HD mice occurred at a stage when they had not developed any neurological symptoms caused by the mutant HD gene. At this stage there was no change in the number of striatal neurons and astrocytes in untreated R6/1 mice, although the striatal volume was decreased by 17%. Moreover, transgenic HD mice had normal striatal levels of NMDA receptors, calbindin D28k (calcium buffer), superoxide dismutase activity (antioxidant enzyme), Bcl-2 (anti-apoptotic protein), heat shock protein 70 (stress-induced anti-apoptotic protein), and citrate synthase activity (mitochondrial enzyme). We propose that the presence of exon 1 of the mutant HD gene induces profound changes in striatal neurons that render these cells resistant to excessive NMDA receptor activation.
Evidence for the role of PrP
C
helix 1 in the hydrophilic seeding of prion aggregates
Authors: Morrissey M. P. , Shakhnovich E. I.
Published: July 2002

Prions are mammalian proteins (PrPs) with a unique pathogenic property: a nonendogenous isoform PrP
Sc
can catalyze conversion of the endogenous PrP
C
isoform into additional PrP
Sc
. In this work, we demonstrate that PrP
C
helix 1 has certain properties (hydrophilicity, charge distribution) that make it unique among all naturally occurring α-helices, and which are indicative of a highly specific model of prion infectivity. The β-nucleation model proposes that PrP
Sc
is an aggregate with a hydrophilic core, consisting of a β-sheet-like arrangement of constituent helix 1 components. It is suggested by using structural arguments, and confirmed by using
charmm
energy calculations, that aggregate formation from two PrP
C
molecules is highly unfavorable, but the addition of chains to an existing aggregate is favorable. The β-nucleation model is shown to be consistent with the prion species-barrier, as well as with infectivity data. Sequence analysis of all known protein structures indicates that PrP is uniquely suited to β-nucleation, in contrast to the many proteins that readily form less favorable (often nonspecific) hydrophobic aggregates.
Hyperactive transposase mutants of the
Himar
1
mariner transposon
Authors: Lampe David J. , Akerley Brian J. , Rubin Eric J. , Mekalanos John J. , Robertson Hugh M.
Published: July 2002

Mariner
-family transposable elements are active in a wide variety of organisms and are becoming increasingly important genetic tools in species lacking sophisticated genetics. The
Himar1
element, isolated from the horn fly,
Haematobia irritans
, is active in
Escherichia coli
when expressed appropriately. We used this fact to devise a genetic screen for hyperactive mutants of
Himar1
transposase that enhance overall transposition from ≈4- to 50-fold as measured in an
E. coli
assay. Purified mutant transposases retain their hyperactivity, although to a lesser degree, in an
in vitro
transposition assay. Mutants like those described herein should enable sophisticated analysis of the biochemistry of
mariner
transposition and should improve the use of these elements as genetic tools, both
in vivo
and
in vitro
.
Slow transport of unpolymerized tubulin and polymerized neurofilament in the squid giant axon
Authors: Galbraith James A. , Reese Thomas S. , Schlief Michelle L. , Gallant Paul E.
Published: July 2002

A major issue in the slow transport of cytoskeletal proteins is the form in which they are transported. We have investigated the possibility that unpolymerized as well as polymerized cytoskeletal proteins can be actively transported in axons. We report the active transport of highly diffusible tubulin oligomers, as well as transport of the less diffusible neurofilament polymers. After injection into the squid giant axon, tubulin was transported in an anterograde direction at an average rate of 2.3 mm/day, whereas neurofilament was moved at 1.1 mm/day. Addition of the metabolic poisons cyanide or dinitrophenol reduced the active transport of both proteins to less than 10% of control values, whereas disruption of microtubules by treatment of the axon with cold in the presence of nocodazole reduced transport of both proteins to ≈20% of control levels. Passive diffusion of these proteins occurred in parallel with transport. The diffusion coefficient of the moving tubulin in axoplasm was 8.6 μm
2
/s compared with only 0.43 μm
2
/s for neurofilament. These results suggest that the tubulin was transported in the unpolymerized state and that the neurofilament was transported in the polymerized state by an energy-dependent nocodazole/cold-sensitive transport mechanism.
The combined absence of the transcription factors Rel and RelA leads to multiple hemopoietic cell defects
Authors: Grossmann Mathis , Metcalf Donald , Merryfull Julie , Beg Amer , Baltimore David , Gerondakis Steve
Published: July 2002

Individual Rel/NF-κB transcription factors, although dispensable for the development and maturation of most hemopoietic cells, are critical regulators of normal immune function. Redundancy among these proteins prompted us to examine the role of Rel and RelA in hemopoiesis by using mice that lack both subunits. Because of the death of double-mutant fetuses at day 13.5 of gestation (E13.5), E12 fetal liver hemopoietic progenitors were used for
in vitro
cultures and for repopulating stem cell studies in lethally irradiated normal recipient mice. Most striking, Rel/RelA-deficient hemopoietic precursors failed to promote the survival of myeloablated mice. This phenotype was associated with several defects including a reduction of spleen colony-forming unit progenitors, impaired erythropoiesis, and a deregulated expansion of granulocytes.
In vitro
progenitor assays also revealed that Rel or RelA serves an antiapoptotic role during monocyte differentiation. Despite the combined loss of Rel and RelA leading to these hemopoietic defects,
c-rel
−/−
rela
−/−
stem cells contributed to the development of all lineages in mice engrafted with double-mutant fetal liver cells and normal bone marrow cells, albeit in a reduced fashion compared with controls. Collectively, these data indicate the loss of Rel and RelA does not appear to affect pluripotent stem cells; rather, Rel and RelA serve redundant functions in regulating differentiation and survival of committed progenitors in multiple hemopoietic lineages.
Abscisic acid signal transduction in guard cells is mediated by phospholipase D activity
Authors: Jacob Tobias , Ritchie Sian , Assmann Sarah M. , Gilroy Simon
Published: July 2002

In guard cells, the plant hormone abscisic acid (ABA) inhibits stomatal opening and induces stomatal closure through the coordinated regulation of ion transport. Despite this central role of ABA in regulating stomatal function, the signal transduction events leading to altered ion fluxes remain incompletely understood. We report that the activity of the enzyme phospholipase D (PLD) transiently increased in guard cell protoplasts at 2.5 and 25 min after ABA application. Treatment of guard cell protoplasts with phosphatidic acid (PtdOH), one of the products of PLD activity, led to an inhibition of the activity of the inward K
+
channel. PtdOH also induced stomatal closure and inhibited stomatal opening when added to epidermal peels. Application of 1-butanol (1-buOH), a selective inhibitor of PtdOH production by PLD, inhibited the increase in PtdOH production elicited by ABA. 1-BuOH treatment also partially prevented ABA-induced stomatal closure and ABA-induced inhibition of stomatal opening. This inhibitory effect of buOH was enhanced by simultaneous application of nicotinamide, an inhibitor of cADP ribose action. These results suggest that in the guard cell, ABA activates the enzyme PLD, which leads to the production of PtdOH. This PtdOH is then involved in triggering subsequent ABA responses of the cell via a pathway operating in parallel to cADP ribose-mediated events.
Correlation of the structural and functional domains in the membrane protein Vpu from HIV-1
Authors: Marassi F. M. , Ma C. , Gratkowski H. , Straus S. K. , Strebel K. , Oblatt-Montal M. , Montal M. , Opella S. J.
Published: July 2002

Vpu is an 81-residue membrane protein encoded by the HIV-1 genome. NMR experiments show that the protein folds into two distinct domains, a transmembrane hydrophobic helix and a cytoplasmic domain with two in-plane amphipathic α-helices separated by a linker region. Resonances in one-dimensional solid-state NMR spectra of uniformly
15
N labeled Vpu are clearly segregated into two bands at chemical shift frequencies associated with NH bonds in a transmembrane α-helix, perpendicular to the membrane surface, and with NH bonds in the cytoplasmic helices parallel to the membrane surface. Solid-state NMR spectra of truncated Vpu
2–51
(residues 2–51), which contains the transmembrane α-helix and the first amphipathic helix of the cytoplasmic domain, and of a construct Vpu
28–81
(residues 28–81), which contains only the cytoplasmic domain, support this structural model of Vpu in the membrane. Full-length Vpu (residues 2–81) forms discrete ion-conducting channels of heterogeneous conductance in lipid bilayers. The most frequent conductances were 22 ± 3 pS and 12 ± 3 pS in 0.5 M KCl and 29 ± 3 pS and 12 ± 3 pS in 0.5 M NaCl. In agreement with the structural model, truncated Vpu
2–51
, which has the transmembrane helix, forms discrete channels in lipid bilayers, whereas the cytoplasmic domain Vpu
28–81
, which lacks the transmembrane helix, does not. This finding shows that the channel activity is associated with the transmembrane helical domain. The pattern of channel activity is characteristic of the self-assembly of conductive oligomers in the membrane and is compatible with the structural and functional findings.
Specific therapy regimes could lead to long-term immunological control of HIV
Authors: Wodarz Dominik , Nowak Martin A.
Published: July 2002

We use mathematical models to study the relationship between HIV and the immune system during the natural course of infection and in the context of different antiviral treatment regimes. The models suggest that an efficient cytotoxic T lymphocyte (CTL) memory response is required to control the virus. We define CTL memory as long-term persistence of CTL precursors in the absence of antigen. Infection and depletion of CD4+T helper cells interfere with CTL memory generation, resulting in persistent viral replication and disease progression. We find that antiviral drug therapy during primary infection can enable the development of CTL memory. In chronically infected patients, specific treatment schedules, either including deliberate drug holidays or antigenic boosts of the immune system, can lead to a re-establishment of CTL memory. Whether such treatment regimes would lead to long-term immunologic control deserves investigation under carefully controlled conditions.
Elimination of residual metastatic prostate cancer after surgery and adjunctive cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) blockade immunotherapy
Authors: Kwon Eugene D. , Foster Barbara A. , Hurwitz Arthur A. , Madias Chris , Allison James P. , Greenberg Norman M. , Burg Maurice B.
Published: July 2002

Cancer relapse after surgery is a common occurrence, most frequently resulting from the outgrowth of minimal residual disease in the form of metastases. We examined the effectiveness of cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) blockade as an adjunctive immunotherapy to reduce metastatic relapse after primary prostate tumor resection. For these studies, we developed a murine model in which overt metastatic outgrowth of TRAMP-C2 (C2) prostate cancer ensues after complete primary tumor resection. Metastatic relapse in this model occurs reliably and principally within the draining lymph nodes in close proximity to the primary tumor, arising from established metastases present at the time of surgery. Using this model, we demonstrate that adjunctive CTLA-4 blockade administered immediately after primary tumor resection reduces metastatic relapse from 97.4 to 44%. Consistent with this, lymph nodes obtained 2 weeks after treatment reveal marked destruction or complete elimination of C2 metastases in 60% of mice receiving adjunctive anti-CTLA-4 whereas 100% of control antibody-treated mice demonstrate progressive C2 lymph node replacement. Our study demonstrates the potential of adjunctive CTLA-4 blockade immunotherapy to reduce cancer relapse emanating from minimal residual metastatic disease and may have broader implications for improving the capability of immunotherapy by combining such forms of therapy with other cytoreductive measures including surgery.
HIV-1 and T cell dynamics after interruption of highly active antiretroviral therapy (HAART) in patients with a history of sustained viral suppression
Authors: Davey Richard T. , Bhat Niranjan , Yoder Christian , Chun Tae-Wook , Metcalf Julia A. , Dewar Robin , Natarajan Ven , Lempicki Richard A. , Adelsberger Joseph W. , Miller Kirk D. , Kovacs Joseph A. , Polis Michael A. , Walker Robert E. , Falloon Judith , Masur Henry , Gee Dennis , Baseler Michael , Dimitrov Dimiter S. , Fauci Anthony S. , Lane H. Clifford
Published: July 2002

Identifying the immunologic and virologic consequences of discontinuing antiretroviral therapy in HIV-infected patients is of major importance in developing long-term treatment strategies for patients with HIV-1 infection. We designed a trial to characterize these parameters after interruption of highly active antiretroviral therapy (HAART) in patients who had maintained prolonged viral suppression on antiretroviral drugs. Eighteen patients with CD4
+
T cell counts ≥ 350 cells/μl and viral load below the limits of detection for ≥1 year while on HAART were enrolled prospectively in a trial in which HAART was discontinued. Twelve of these patients had received prior IL-2 therapy and had low frequencies of resting, latently infected CD4 cells. Viral load relapse to >50 copies/ml occurred in all 18 patients independent of prior IL-2 treatment, beginning most commonly during weeks 2–3 after cessation of HAART. The mean relapse rate constant was 0.45 (0.20 log
10
copies) day
−1
, which was very similar to the mean viral clearance rate constant after drug resumption of 0.35 (0.15 log
10
copies) day
−1
(
P
= 0.28). One patient experienced a relapse delay to week 7. All patients except one experienced a relapse burden to >5,000 RNA copies/ml.
Ex vivo
labeling with BrdUrd showed that CD4 and CD8 cell turnover increased after withdrawal of HAART and correlated with viral load whereas lymphocyte turnover decreased after reinitiation of drug treatment. Virologic relapse occurs rapidly in patients who discontinue suppressive drug therapy, even in patients with a markedly diminished pool of resting, latently infected CD4
+
T cells.
Estradiol repression of tumor necrosis factor-α transcription requires estrogen receptor activation function-2 and is enhanced by coactivators
Authors: An Jinping , Ribeiro Ralff C. J. , Webb Paul , Gustafsson Jan-Åke , Kushner Peter J. , Baxter John D. , Leitman Dale C.
Published: July 2002

The tumor necrosis factor-α (TNF-α) promoter was used to explore the molecular mechanisms of estradiol (E
2
)-dependent repression of gene transcription. E
2
inhibited basal activity and abolished TNF-α activation of the TNF-α promoter. The E
2
-inhibitory element was mapped to the −125 to −82 region of the TNF-α promoter, known as the TNF-responsive element (TNF-RE). An AP-1-like site in the TNF-RE is essential for repression activity. Estrogen receptor (ER) β is more potent than ERα at repressing the −1044 TNF-α promoter and the TNF-RE upstream of the herpes simplex virus thymidine kinase promoter, but weaker at activating transcription through an estrogen response element. The activation function-2 (AF-2) surface in the ligand-binding domain is required for repression, because anti-estrogens and AF-2 mutations impair repression. The requirement of the AF-2 surface for repression is probably due to its capacity to recruit p160 coactivators or related coregulators, because overexpressing the coactivator glucocorticoid receptor interacting protein-1 enhances repression, whereas a glucocorticoid receptor interacting protein-1 mutant unable to interact with the AF-2 surface is ineffective. Furthermore, receptor interacting protein 140 prevents repression by ERβ, probably by interacting with the AF-2 surface and blocking the binding of endogenous coactivators. These studies demonstrate that E
2
-mediated repression requires the AF-2 surface and the participation of coactivators or other coregulatory proteins.
Targeted disruption of gene function in
Drosophila
by RNA interference (RNA-i): A role for
nautilus
in embryonic somatic muscle formation
Authors: Misquitta Leonie , Paterson Bruce M.
Published: July 2002

The expression of the MyoD gene homolog,
nautilus
(nau), in the
Drosophila
embryo defines a subset of
mesodermal cells known as the muscle “pioneer” or “founder”
cells. These cells are thought to establish the future muscle pattern
in each hemisegment. Founders appear to recruit fusion-competent
mesodermal cells to establish a particular muscle fiber type. In
support of this concept every somatic muscle in the embryo is
associated with one or more
nautilus
-positive cells.
However, because of the lack of known (isolated)
nautilus
mutations, no direct test of the founder cell
hypothesis has been possible. We now have utilized toxin ablation and
genetic interference by double-stranded RNA (RNA interference or RNA-i)
to determine both the role of the
nautilus
-expressing
cells and the
nautilus
gene, respectively, in embryonic
muscle formation. In the absence of
nautilus
-expressing
cells muscle formation is severely disrupted or absent. A similar
phenotype is observed with the elimination of the
nautilus
gene product by genetic interference upon
injection of
nautilus
double-stranded RNA. These results
define a crucial role for
nautilus
in embryonic muscle
formation. The application of RNA interference to a variety of known
Drosophila
mutations as controls gave phenotypes
essentially indistinguishable from the original mutation. RNA-i
provides a powerful approach for the targeted disruption of a given
genetic function in
Drosophila
.
Native display of complete foreign protein domains on the surface of hepatitis B virus capsids
Authors: Kratz Peter A. , Böttcher Bettina , Nassal Michael
Published: July 2002

The nucleocapsid of hepatitis B virus (HBV), or HBcAg, is a highly symmetric structure formed by multiple dimers of a single core protein that contains potent T helper epitopes in its 183-aa sequence. Both factors make HBcAg an unusually strong immunogen and an attractive candidate as a carrier for foreign epitopes. The immunodominant c/e1 epitope on the capsid has been suggested as a superior location to convey high immunogenicity to a heterologous sequence. Because of its central position, however, any c/e1 insert disrupts the core protein’s primary sequence; hence, only peptides, or rather small protein fragments seemed to be compatible with particle formation. According to recent structural data, the epitope is located at the tips of prominent surface spikes formed by the very stable dimer interfaces. We therefore reasoned that much larger inserts might be tolerated, provided the individual parts of a corresponding fusion protein could fold independently. Using the green fluorescent protein (GFP) as a model insert, we show that the chimeric protein efficiently forms fluorescent particles; hence, all of its structurally important parts must be properly folded. We also demonstrate that the GFP domains are surface-exposed and that the chimeric particles elicit a potent humoral response against native GFP. Hence, proteins of at least up to 238 aa can be natively displayed on the surface of HBV core particles. Such chimeras may not only be useful as vaccines but may also open the way for high resolution structural analyses of nonassembling proteins by electron microscopy.
Poly(ADP-ribose) polymerase-deficient mice are protected from streptozotocin-induced diabetes
Authors: Pieper Andrew A. , Brat Daniel J. , Krug David K. , Watkins Crystal C. , Gupta Alok , Blackshaw Seth , Verma Ajay , Wang Zhao-Qi , Snyder Solomon H.
Published: July 2002

Streptozotocin (STZ) selectively destroys insulin-producing beta islet cells of the pancreas providing a model of type I diabetes. Poly(ADP-ribose) polymerase (PARP) is a nuclear enzyme whose overactivation by DNA strand breaks depletes its substrate NAD
+
and then ATP, leading to cellular death from energy depletion. We demonstrate DNA damage and a major activation of PARP in pancreatic islets of STZ-treated mice. These mice display a 500% increase in blood glucose and major pancreatic islet damage. In mice with homozygous targeted deletion of PARP (PARP −/−), blood glucose and pancreatic islet structure are normal, indicating virtually total protection from STZ diabetes. Partial protection occurs in PARP +/− animals. Thus, PARP activation may participate in the pathophysiology of type I diabetes, for which PARP inhibitors might afford therapeutic benefit.
Spatial and temporal regulation of gap junction connexin43 in vascular endothelial cells exposed to controlled disturbed flows
in vitro
Authors: DePaola Natacha , Davies Peter F. , Pritchard William F. , Florez Lucio , Harbeck Nadeene , Polacek Denise C.
Published: July 2002

Hemodynamic regulation of the endothelial gap junction protein connexin43 (Cx43) was studied in a model of controlled disturbed flows
in vitro
. Cx43 mRNA, protein expression, and intercellular communication were mapped to spatial variations in fluid forces. Hemodynamic features of atherosclerotic lesion-prone regions of the vasculature (flow separation and recirculation) were created for periods of 5, 16, and 30 h, with laminar shear stresses ranging between 0 and 13.5 dynes/cm
2
. Within 5 h, endothelial Cx43 mRNA expression was increased in all cells when compared with no-flow controls, with highest levels (up to 6- to 8-fold) expressed in regions of flow recirculation corresponding to high shear stress gradients. At 16 h, Cx43 mRNA expression remained elevated in regions of flow disturbance, whereas in areas of fully developed, undisturbed laminar flow, Cx43 expression returned to control levels. In all flow regions, typical punctate Cx43 immunofluorescence at cell borders was disrupted by 5 h. After 30 h of flow, disruption of gap junctions persisted in cells subjected to flow separation and recirculation, whereas regions of undisturbed flow were substantially restored to normal. These expression differences were reflected in sustained inhibition of intercellular communication (dye transfer) throughout the zone of disturbed flow (84.2 and 68.4% inhibition at 5 and 30 h, respectively); in contrast, communication was fully reestablished by 30 h in cells exposed to undisturbed flow. Up-regulation of Cx43 transcripts, sustained disorganization of Cx43 protein, and impaired communication suggest that shear stress gradients in regions of disturbed flow regulate intercellular communication through the expression and function of Cx43.
Biochemical evolution III: Polymerization on organophilic silica-rich surfaces, crystal–chemical modeling, formation of first cells, and geological clues
Authors: Smith Joseph V. , Arnold Frederick P. , Parsons Ian , Lee Martin R.
Published: July 2002

Catalysis at organophilic silica-rich surfaces of zeolites and
feldspars might generate replicating biopolymers from simple chemicals
supplied by meteorites, volcanic gases, and other geological sources.
Crystal–chemical modeling yielded packings for amino acids neatly
encapsulated in 10-ring channels of the molecular sieve
silicalite-ZSM-5-(mutinaite)
. Calculation of binding and
activation energies for catalytic assembly into polymers is progressing
for a chemical composition with one catalytic Al–OH site per 25
neutral Si tetrahedral sites. Internal channel intersections and
external terminations provide special stereochemical features suitable
for complex organic species. Polymer migration along nano/micrometer
channels of ancient weathered feldspars, plus exploitation of
phosphorus and various transition metals in entrapped apatite and other
microminerals, might have generated complexes of replicating catalytic
biomolecules, leading to primitive cellular organisms. The first cell
wall might have been an internal mineral surface, from which the cell
developed a protective biological cap emerging into a nutrient-rich
“soup.” Ultimately, the biological cap might have expanded into a
complete cell wall, allowing mobility and colonization of energy-rich
challenging environments. Electron microscopy of honeycomb channels
inside weathered feldspars of the Shap granite (northwest England) has
revealed modern bacteria, perhaps indicative of Archean ones. All known
early rocks were metamorphosed too highly during geologic time to
permit simple survival of large-pore zeolites, honeycombed feldspar,
and encapsulated species. Possible microscopic clues to the proposed
mineral adsorbents/catalysts are discussed for planning of systematic
study of black cherts from weakly metamorphosed Archaean sediments.