bluuebunny/crossref_metadata_2025 huggingface dataset:
Proposed active site domain in estrogen sulfotransferase as determined by mutational analysis.
Authors: Driscoll W J , Komatsu K , Strott C A
Published: May 2006

Point mutations were selectively introduced into a cDNA for guinea pig estrogen sulfotransferase (gpEST); each construct was then expressed in Chinese hamster ovary K1 cells. The molecular site chosen for study is a conserved GXXGXXK sequence that resembles the P-loop-type nucleotide-binding motif for ATP- and GTP-binding proteins and is located near the C terminus of all steroid and phenol(aryl) sulfotransferases for which the primary structures are known. Preliminary experiments demonstrated that the GXXGXXK motif is essential for binding the activated sulfonate donor 3'-phosphoadenosine 5'-phosphosulfate (PAPS). The present study was undertaken to ascertain the relative importance of each individual residue of the motif. While the mutation of a single motif residue had little effect on the interaction between gpEST and PAPS as determined by kinetic analysis and photoaffinity labeling, the mutation of any two residues in concert resulted in an approximate 10-fold increase in the Km for PAPS and reduced photoaffinity labeling. The mutation of all three motif residues resulted in an inactive enzyme and complete loss of photoaffinity labeling. Interestingly, several mutants also displayed a striking effect on the Km for the steroid substrate; double mutants, again, demonstrated greater perturbations (8- to 28-fold increase) than did single mutants. Unexpectedly, whereas the mutation of nonmotif residues had a negligible effect on the Km for PAPS, a marked increase in the Km for the estrogen substrate ( > 30-fold) was noted. On the basis of these findings, it is concluded that the sequence GISGDWKN within the C-terminal domain of gpEST represents a critical component of the active site.
Latent N-methyl-D-aspartate receptors in the recurrent excitatory pathway between hippocampal CA1 pyramidal neurons: Ca(2+)-dependent activation by blocking A1 adenosine receptors.
Authors: Klishin A , Tsintsadze T , Lozovaya N , Krishtal O
Published: May 2006

When performed at increased external [Ca2+]/[Mg2+] ratio (2.5 mM/0.5 mM), temporary block of A1 adenosine receptors in hippocampus [by 8-cyclopentyltheophylline (CPT)] leads to a dramatic and irreversible change in the excitatory postsynaptic current (EPSC) evoked by Schaffer collateral/commissural (SCC) stimulation and recorded by in situ patch clamp in CA1 pyramidal neurons. The duration of the EPSC becomes stimulus dependent, increasing with increase in stimulus strength. The later occurring component of the EPSC is carried through N-methyl-D-aspartate (NMDA) receptor-operated channels but disappears under either the NMDA antagonist 2-amino-5-phosphonovaleric acid (APV) or the non-NMDA antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). These findings indicate that the late component of the SCC-evoked EPSC is polysynaptic: predominantly non-NMDA receptor-mediated SCC inputs excite CA1 neurons that recurrently excite each other by predominantly NDMA receptor-mediated synapses. These recurrent connections are normally silent but become active after CPT treatment, leading to enhancement of the late component of the EPSC. The activity of these connections is maintained for at least 2 hr after CPT removal. When all functional NMDA receptors are blocked by dizocilpine maleate (MK-801), subsequent application of CPT leads to a partial reappearance of NMDA receptor-mediated EPSCs evoked by SCC stimulation, indicating that latent NMDA receptors are recruited. Altogether, these findings indicate the existence of a powerful system of NMDA receptor-mediated synaptic contacts in SCC input to hippocampal CA1 pyramidal neurons and probably also in reciprocal connections between these neurons, which in the usual preparation are kept latent by activity of A1 receptors.
Learning about categories in the absence of memory.
Authors: Squire L R , Knowlton B J
Published: May 2006

A fundamental question about memory and cognition concerns how information is acquired about categories and concepts as the result of encounters with specific instances. We describe a profoundly amnesic patient (E.P.) who cannot learn and remember specific instances--i.e., he has no detectable declarative memory. Yet after inspecting a series of 40 training stimuli, he was normal at classifying novel stimuli according to whether they did or did not belong to the same category as the training stimuli. In contrast, he was unable to recognize a single stimulus after it was presented 40 times in succession. These findings demonstrate that the ability to classify novel items, after experience with other items in the same category, is a separate and parallel memory function of the brain, independent of the limbic and diencephalic structures essential for remembering individual stimulus items (declarative memory). Category-level knowledge can be acquired implicitly by cumulating information from multiple training examples in the absence of detectable conscious memory for the examples themselves.
In situ detection, by spin trapping, of hydroxyl radical markers produced from ionizing radiation in the tumor of a living mouse.
Authors: Halpern H J , Yu C , Barth E , Peric M , Rosen G M
Published: May 2006

Hydroxyl radicals are thought to be responsible for the toxicity associated with ionizing radiation in tissues. Measurements of hydroxyl radicals generated by ionizing radiation in cellular systems have failed thus far to elucidate higher-level homeostatic responses to this and other reactive oxygen species. Careful assessment of prior indirect hydroxyl radical assays in living tissues indicates that they are prone to a variety of artifacts, making all but the most qualitative relationships difficult to establish. This paper describes the detection of hydroxyl radicals produced during radiation in the leg tumor of a living mouse, where the free radicals evolve; detection uses low-frequency electron paramagnetic resonance in combination with in vivo spin trapping. To our knowledge, this is the first report of such a direct measurement of free radical production in the tissues of a living animals.
CD18 (beta 2 leukocyte integrin) promoter requires PU.1 transcription factor for myeloid activity.
Authors: Rosmarin A G , Caprio D , Levy R , Simkevich C
Published: May 2006

Normal cellular differentiation is linked to tightly regulated gene transcription. However, the DNA elements and trans-acting factors that regulate transcription in myeloid cells are poorly defined. CD18, the beta chain of the leukocyte integrins, is transcriptionally regulated during myeloid differentiation. The CD18 promoter is active after transfection into myeloid cells. We demonstrate that a region of the CD18 promoter that contains two binding sites for the PU.1 transcription factor is required for activity in myeloid cells. These sites are bound by in vitro translated PU.1 and by PU.1 from myeloid nuclear extracts. Mutagenesis of these sites abrogates binding by PU.1 and substantially decreases promoter activity in myeloid cells. Thus, the leukocyte-specific transcription factor PU.1 is required for myeloid activity of CD18.
A targeted mutation in the mouse E-cadherin gene results in defective preimplantation development.
Authors: Riethmacher D , Brinkmann V , Birchmeier C
Published: May 2006

The Ca(2+)-dependent cell adhesion molecule E-cadherin functions in the establishment and maintenance of epithelial cell morphology during embryogenesis and adulthood. Downregulation or complete shut-down of E-cadherin expression and mutation of the gene are observed during the progression of tumors of epithelial origin (carcinomas) and correlate with the metastatic potential. We have introduced a targeted mutation into the E-cadherin gene by homologous recombination in mouse embryonic stem cells. The mutation removes E-cadherin sequences essential for Ca2+ binding and for adhesive function. These embryonic stem cells were used to generate mice carrying the mutation. Heterozygous mutant animals appear normal and are fertile. However, the homozygous mutation is not compatible with life: E-cadherin -/- embryos show severe abnormalities before implantation. Particularly, the adhesive cells of the morula dissociate shortly after compaction has occurred, and their morphological polarization is then destroyed. Interestingly, the blastomers are still able to form desmosomes and tight junctions at sites of distorted cell-cell contact. Thus, maternal E-cadherin suffices for initial compaction of the morula but not for further preimplantation development to occur.
Intra- and intermolecular spreading of autoimmunity involving the nuclear self-antigens La (SS-B) and Ro (SS-A).
Authors: Topfer F , Gordon T , McCluskey J
Published: May 2006

We have tested the extent of immune self-tolerance to the ubiquitously expressed nuclear/cytoplasmic autoantigens La (SS-B) and Ro (SS-A) in healthy, nonautoimmune mice. Immunization of mice with recombinant mouse La resulted in a specific, isotype-switched autoantibody response, which was initially directed toward the La C subfragment (aa 111-242) but rapidly spread to involve the La A (aa 1-107) and La F (aa 243-345) regions of the La antigen. Intramolecular spreading of the anti-La antibody response was further demonstrated by the appearance of autoantibodies to multiple, nonoverlapping antigenic regions of La, after immunization of mice with the 107-aa La A subfragment. Moreover, immunization of mice with recombinant mouse or human La also elicited specific anti-60-kDa Ro IgG antibodies in all strains tested. Mice immunized with 60-kDa Ro produced a high titer anti-Ro antibody response, which was also associated with intermolecular spreading, resulting in the specific appearance of anti-La autoantibodies. These findings show that the development of autoantibodies to multiple components of the La/Ro ribonucleoprotein complex may follow initiation of immunity to a single component. In addition, the data reveal the incomplete nature of immune tolerance to La and Ro despite their endogenous expression in all nucleated cells. These observations are likely to account for the coexistence of anti-La/Ro antibodies in autoimmune disease and suggest a general explanation for the appearance of mixed autoantibody patterns in systemic autoimmune disorders.
Characterization of the genomic breakpoint and chimeric transcripts in the EWS-WT1 gene fusion of desmoplastic small round cell tumor.
Authors: Gerald W L , Rosai J , Ladanyi M
Published: May 2006

Desmoplastic small round cell tumor is a recently recognized distinctive tumor shown to be associated with a recurrent translocation, t(11;22)(p13;q12), and rearrangement of the genes for Ewing sarcoma (EWS) and Wilms tumor (WT1). A genomic DNA fragment containing the EWS-WT1 gene fusion has been isolated from a desmoplastic small round cell tumor, and the breakpoint has been characterized. The breakpoints involve the intron between EWS exons 7 and 8 and the intron between WT1 exons 7 and 8. Chimeric transcripts corresponding to the fusion gene were detected in four of six cases studied. Analysis of these transcripts show an in-frame fusion of RNA encoding the amino-terminal domain of EWS to both alternatively spliced forms of the last three zinc fingers of the DNA-binding domain of WT1. Desmoplastic small round cell tumor represents the third tumor type associated with translocation of EWS and the first tumor associated with consistent translocation of WT1. The chimeric products are predicted to modulate transcription at WT1 target sites and contribute to development of this unique tumor.
Stringent control and growth-rate-dependent control have nonidentical promoter sequence requirements.
Authors: Josaitis C A , Gaal T , Gourse R L
Published: May 2006

Escherichia coli uses at least two regulatory systems, stringent control and growth-rate-dependent control, to adjust rRNA output to amino acid availability and the steady-state growth rate, respectively. We examined transcription from rrnB P1 promoters containing or lacking the cis-acting UP element and FIS protein binding sites after amino acid starvation. The "core promoter" responds to amino acid starvation like the full-length wild-type promoter; thus, neither the UP element nor FIS plays a role in stringent control. To clarify the relationship between growth-rate-dependent regulation and stringent control, we measured transcription from growth-rate-independent promoters during amino acid starvation. Four rrnB P1 mutants defective for growth-rate control and two other growth-rate-independent promoters (rrnB P2 and pS10) still displayed stringent regulation. Thus, the two systems have different promoter determinants, consistent with the idea that they function by different mechanisms. Two mutations disrupted stringent control of rrnB P1: (i) a multiple base change in the "discriminator" region between the -10 hexamer and the transcription start site and (ii) a double substitution making the promoter resemble the E sigma 70 consensus promoter. These results have important implications for the mechanisms of both stringent control and growth-rate-dependent control of rRNA transcription.
Quinoxalines block the mechanism of directional selectivity in ganglion cells of the rabbit retina.
Authors: Cohen E D , Miller R F
Published: May 2006

Direction selectivity is a receptive field property displayed by neurons throughout the visual system. Previous experiments have concentrated on the role of lateral connections that use gamma-aminobutyric acid and acetylcholine. We have examined the role of excitatory amino acid receptors on direction-selective ganglion cell function in the rabbit retina. Application of the quinoxalines, a group of kainate/alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor antagonists, selectively blocked the directional-selectivity mechanism, leaving cells responsive to both directions of movement. In contrast, direction selectivity was unaffected by N-methyl-D-aspartate receptor antagonists or L-2-amino-4-phosphonobutyric acid. Large reductions in acetylcholine release by starburst amacrine cells appear to parallel losses of direction selectivity observed in the quinoxalines. These results shed additional insights into the mechanism of direction selectivity.
Expression cloning of cardiotrophin 1, a cytokine that induces cardiac myocyte hypertrophy.
Authors: Pennica D , King K L , Shaw K J , Luis E , Rullamas J , Luoh S M , Darbonne W C , Knutzon D S , Yen R , Chien K R
Published: May 2006

Heart failure continues to be a leading cause of mortality worldwide. A hallmark of this disease is dilated cardiac hypertrophy, which is accompanied by a reactivation of genes expressed in fetal heart development. Reasoning that fetal or embryonic growth factors may mediate the onset of cardiac hypertrophy, we have coupled expression cloning with an embryonic stem cell-based model of cardiogenesis to isolate a 21.5-kDa protein, cardiotrophin 1, that potently induces cardiac myocyte hypertrophy in vitro. Amino acid similarity data indicate that cardiotrophin 1 is a member of the leukemia inhibitory factor/ciliary neurotrophic factor/oncostatin M/interleukin 6/interleukin 11 family of cytokines. Several members of this family that are known to signal through the transmembrane protein gp130 stimulate cardiac myocyte hypertrophy, like cardiotrophin 1, suggesting that the gp130 signaling pathway may play a role in cardiac hypertrophy. A 1.4-kb cardiotrophin 1 mRNA is expressed in the heart and several other mouse tissues.
Retrovirus-mediated transfer of an angiotensin type I receptor (AT1-R) antisense sequence decreases AT1-Rs and angiotensin II action in astroglial and neuronal cells in primary cultures from the brain.
Authors: Lu D , Yu K , Raizada M K
Published: May 2006

The AT1-R has been implicated in many cellular and physiological actions of angiotensin II (AII) in the brain. A retrovirus vector (LNSV) containing an AT1B-R antisense sequence (AT1B-AS) (termed LNSV-AT1B-AS) was constructed and used to determine the feasibility of using viral-mediated gene transfer to control AT1-Rs and AII actions in astroglial and neuronal cells in primary cultures from rat brain. Briefly, a 1.26-kb antisense sequence corresponding to nt -132 to +1128 of AT1-R cDNA was cloned into the LNSV vector, the vector was transfected into PA317 cells, and transfected cells were selected in G418. Incubation of brain cells with culture medium containing LNSV-AT1B-AS viral particles showed that AT1B-AS was integrated into the genome and transcribed in brain cells. This was associated with a significant decrease in AT1-Rs and in the AII-stimulated increase of c-fos mRNA, a measure of AT1-R function. These observations show that the AT1B-AS gene can be transferred into astroglial cells in culture by LNSV and that such a transfer inhibits AT1-Rs and the AII stimulation of cellular activities. In addition, the usefulness of this approach to study AII-dependent pathophysiology in primary neuronal cultures from brain, in particular, is established.
Identification of galectin-3 as a factor in pre-mRNA splicing.
Authors: Dagher S F , Wang J L , Patterson R J
Published: May 2006

Galectin-3 (M(r) approximately 35,000) is a galactose/lactose-specific lectin found in association with ribonucleoprotein complexes in many animal cells. Cell-free-splicing assays have been carried out to study the requirement for galectin-3 in RNA processing by HeLa cell nuclear extracts by using 32P-labeled MINX as the pre-mRNA substrate. Addition of saccharides that bind galectin-3 with high affinity inhibited product formation in the splicing assay, while addition of carbohydrates that do not bind to the lectin did not inhibit product formation. Nuclear extracts depleted of galectin-3 by affinity adsorption on a lactose-agarose column were deficient in splicing activity. Extracts subjected to parallel adsorption on control cellobiose-agarose retained splicing activity. The activity of the galectin-3-depleted extract could be reconstituted by the addition of purified recombinant galectin-3, whereas the addition of other lectins, either with a similar saccharide binding specificity (soybean agglutinin) or with a different specificity (wheat germ agglutinin), did not restore splicing activity. The formation of splicing complexes was also sensitive to galectin-3 depletion and reconstitution. Together, these results define a requirement for galectin-3 in pre-mRNA splicing and identify it as a splicing factor.
Contribution of cotranslational folding to the rate of formation of native protein structure.
Authors: Fedorov A N , Baldwin T O
Published: May 2006

To compare the process of protein folding in the cell with refolding following denaturation in vitro, we have investigated and compared the kinetics of renaturation of a full-length protein upon dilution from concentrated urea with the rate of folding in the course of biosynthesis. Formation of enzymatically active bacterial luciferase, an alpha beta heterodimer, occurred 2 min after completion of beta-subunit synthesis in an Escherichia coli cell-free system. Renaturation of urea-denatured beta subunit, either in the presence of the cell-free protein synthesis system or in buffer solutions, proceeded more slowly. Cellular components present in the cell-free protein synthesis system slightly accelerated the rate of refolding of urea-unfolded beta subunit. The results indicate that the luciferase beta subunit begins the folding process cotranslationally and that cotranslational folding contributes to the rapid formation of the native structure in the cell.
Two closely linked but separable promoters for human neuronal nitric oxide synthase gene transcription.
Authors: Xie J , Roddy P , Rife T K , Murad F , Young A P
Published: May 2006

In this report we demonstrate that the human cerebellum contains neuronal nitric oxide synthase (nNOS) mRNAs with two distinct 5'-untranslated regions that are encoded through use of closely linked but separate promoters. nNOS cDNA clones were shown to contain different 5' terminal exons spliced to a common exon 2. Genomic cloning and sequence analysis demonstrate that the unique exons are positioned within 300 bp of each other but separated from exon 2 by an intron that is at least 20 kb in length. A CpG island engulfs the downstream 5'-terminal exon. In contrast, most of the upstream exon resides outside of this CpG island. Interestingly, the upstream exon includes a GT dinucleotide repeat. A fusion gene with a 414-bp nNOS genomic fragment that includes a portion of the upstream 5'-terminal exon and its immediate 5'-flanking DNA is expressed in transfected HeLa cells. Also expressed is a fusion gene that contains the luciferase reporter under transcriptional control by a 308-bp genomic fragment that includes the region separating both 5'-terminal exons. These results indicate that expression of these exons is subject to transcriptional control by separate promoters. However, the proximity of these promoters raise the possibility that complex interactions may be involved in regulating nNOS gene expression at these sites.
Superoxide dismutase is an abundant component in cell bodies, dendrites, and axons of motor neurons and in a subset of other neurons.
Authors: Pardo C A , Xu Z , Borchelt D R , Price D L , Sisodia S S , Cleveland D W
Published: May 2006

Mutation in superoxide dismutase 1 (SOD1), a Cu/Zn enzyme that removes oxygen radicals and protects against oxidative injury, has been implicated in some cases of familial amyotrophic lateral sclerosis (FALS). As a first approach to examining the mechanism(s) through which these mutations cause specific degeneration of motor neurons, we have used immunocytochemistry to identify the distribution of SOD1 in populations of cells in the peripheral and central nervous systems. In the spinal cord, intense SOD1 immunoreactivity was present in motor neurons, interneurons, and substantia gelatinosa. In motor neurons, SOD1 immunoreactivity was abundant in perikarya, dendrites, and axons; most of this activity appeared to be free in the cytoplasm, although a portion was associated with membranous vesicles, presumably peroxisomes. Since a variety of central nervous system neurons, including pyramidal cells in cerebral cortex and neurons of the CA3 and CA4 sectors of the hippocampus, showed high immunoreactivity but are unaffected in ALS, the apparent abundance of SOD1 does not predict vulnerability of neurons to mutations in SOD1. Rather, SOD1 accumulates in many neuronal populations but is particularly abundant in motor neurons. Consistent with recent studies of FALS-linked SOD1 mutations in vitro and in transgenic mice, our findings offer further support for the view that the mutations confer a gain of adverse function. In this view, high, rather than limiting, levels of SOD1 may place motor neurons selectively at risk in FALS.
Early high-affinity neutralizing anti-viral IgG responses without further overall improvements of affinity.
Authors: Roost H P , Bachmann M F , Haag A , Kalinke U , Pliska V , Hengartner H , Zinkernagel R M
Published: May 2006

Affinity maturation of IgG antibodies in adaptive immune responses is a well-accepted mechanism to improve effector functions of IgG within 2 weeks to several months of antigen encounter. This concept has been defined mainly for IgG responses against chemically defined haptens. We have evaluated this concept in a viral system and analyzed neutralizing IgG antibody responses against vesicular stomatitis virus (a close relative of rabies virus) with a panel of monoclonal antibodies obtained early (day 6 or 12) and late (day 150) after hyperimmunization. These neutralizing IgG antibodies recognize a single major antigenic site with high affinities (Ka of 10(8)-10(10) liter.mol-1) and with rapid on-rates already on day 6 of a primary response and with no evidence for further antigen dose- and time-dependent overall improvement of affinity. This type of IgG response is probably representative for viruses or bacterial toxins which are crucially controlled by neutralizing antibodies.
Real time imaging of transcriptional activity in live mouse preimplantation embryos using a secreted luciferase.
Authors: Thompson E M , Adenot P , Tsuji F I , Renard J P
Published: May 2006

Study of gene expression kinetics during preimplantation mammalian development is difficult because of the limited amount of material and the usually destructive, static nature of molecular analyses. We describe continuous, noninvasive monitoring of gene expression in preimplantation embryos by using a secreted luminescent reporter, Vargula luciferase. Transgene expression profiles were followed by assaying aliquots of culture medium or by direct visualization of Vargula luciferase secretion from living embryos in real time through photon imaging. With this approach, it is possible to observe epigenetic modulations of gene expression and to link this over time to the developmental capacity of individual embryos. In addition, by developing a strategy where expression from integrated transgenes is enhanced relative to that from nonintegrated DNA, we provide evidence that rapid detection of transgene integration prior to the blastocyst stage should be possible. Thus, imaging of Vargula luciferase secretion may also be useful in the early screening of embryos, for example, in the production of transgenic livestock.
Development of anti-p185HER2 immunoliposomes for cancer therapy.
Authors: Park J W , Hong K , Carter P , Asgari H , Guo L Y , Keller G A , Wirth C , Shalaby R , Kotts C , Wood W I
Published: May 2006

The product of the HER2 protooncogene, p185HER2, represents an attractive target for cancer immunotherapies. We have prepared anti-p185HER2 immunoliposomes in which Fab' fragments of a humanized anti-p185HER2 monoclonal antibody with antiproliferative properties (rhuMAb-HER2) were conjugated to either conventional or sterically stabilized liposomes. These immunoliposomes bind specifically to p185HER2-overexpressing breast cancer cells (SK-BR-3 and BT-474). High-affinity binding of anti-p185HER2 immunoliposomes is comparable to that of free rhuMAbHER2-Fab' or the intact antibody. Empty immunoliposomes inhibit the culture growth of p185HER2-overexpressing breast cancer cells, and this antiproliferative effect is superior to that of free rhuMAbHER2-Fab', indicating that liposomal anchoring of these anti-p185HER2 Fab' fragments enhances their biological activity. Efficient internalization of anti-p185HER2 immunoliposomes, demonstrated by light and electron microscopy, occurs by receptor-mediated endocytosis via the coated pit pathway and also possibly by membrane fusion. Doxorubicin-loaded anti-p185HER2 immunoliposomes are markedly and specifically cytotoxic against p185HER2-overexpressing tumor cells in vitro. Anti-p185HER2 immunoliposomes administered in vivo in Scid mice bearing human breast tumor (BT-474) xenografts can deliver doxorubicin to tumors. These results indicate that anti-p185HER2 immunoliposomes are a promising therapeutic vehicle for the treatment of p185HER2-overexpressing human cancers.
Evidence that carcinogenesis involves an imbalance between epigenetic high-frequency initiation and suppression of promotion.
Authors: Kamiya K , Yasukawa-Barnes J , Mitchen J M , Gould M N , Clifton K H
Published: May 2006

Evidence is presented in support of the hypothesis that cancer development depends on an imbalance between highly frequent epigenetic initiation and suppression of promotion of the initiated cells. When irradiated clonogenic mammary epithelial cells are transplanted and hormonally stimulated, they give rise to clonal glandular structures within which carcinomas may arise. In the current study, the cancer incidence in grafts of approximately 13 7-Gy-irradiated clonogens per site indicated that at least 1 of approximately 95 clonogens was radiogenically initiated. A similar initiation frequency had been seen in grafts of approximately 5 methylnitrosourea (MNU)-treated clonogens. Such initiation is thus far more frequent than specific locus mutations. In sites grafted with larger cell inocula, cancer incidences per clonogen were suppressed inversely as the numbers of irradiated or MNU-treated clonogens per graft increased. Addition of unirradiated cells to small irradiated graft inocula also suppressed progression. Radiation and MNU thus produce quantitatively, and perhaps qualitatively, similar carcinogenesis-related sequelae in mammary clonogens.
Leukemia inhibitory factor protects against experimental lethal Escherichia coli septic shock in mice.
Authors: Waring P M , Waring L J , Billington T , Metcalf D
Published: May 2006

Leukemia inhibitory factor (LIF) has recently been associated with septic shock in humans. In this study we sought to determine, in mice, the role of LIF in septic shock. During sublethal endotoxemia, serum LIF levels, as determined by radio-receptor competition assay, peaked at 2 h and were low (3 ng/ml), whereas in lethal Escherichia coli septic shock serum LIF levels rose progressively (> 30 ng/ml) in the premorbid phase coincident with the development of tissue injury. Single i.v. injections of high doses (up to 50 micrograms per mouse) of recombinant murine LIF had no obvious acute detrimental effects, whereas continued i.p. administration (30 micrograms per mouse per day) for 3-4 days induced a fatal catabolic state without evidence of preceding hemodynamic collapse or shock. Simultaneous or subsequent administration of high doses of LIF had no effect on mortality from sublethal and lethal E. coli septic shock, whereas prior administration conferred significant protection against lethality (P << 0.001 by log-rank test), an effect that was dose and interval dependent. This protective effect resembled endotoxin tolerance and was characterized by suppression of E. coli-induced serum tumor necrosis factor concentration (P < 0.05), reduction in the number of viable bacteria (P < 0.05), and prevention of sepsis-induced tissue injury. These observations suggest that systemic LIF production is part of the host response to both endotoxin and sepsis-induced tissue injury.
Plasma albumin is a potent trigger of calcium signals and DNA synthesis in astrocytes.
Authors: Nadal A , Fuentes E , Pastor J , McNaughton P A
Published: May 2006

Cells in the central nervous system are normally prevented from coming into contact with albumin and other protein components of blood by the existence of a tight blood-brain barrier. Astrocytes and other glial cells proliferate to form glial scars when the blood-brain barrier is breached. In this report we show that albumin is an important blood component responsible for inducing astrocyte proliferation. Albumin also generates maintained trains of calcium spikes in astrocytes. Neither activity depends on blood coagulation, as albumins from both serum and plasma are approximately equally effective. Methanol extraction of albumin abolishes both actions, and recombination of the methanol-extracted factor with extracted albumin restores full activity indistinguishable from that of native albumin. The factor is sensitive to lipase, and the solvent extraction profile is that of a polar lipid.
Axon-induced mitogenesis of human Schwann cells involves heregulin and p185erbB2.
Authors: Morrissey T K , Levi A D , Nuijens A , Sliwkowski M X , Bunge R P
Published: May 2006

The ability of sensory axons to stimulate Schwann cell proliferation by contact was established in the 1970s. Although the mitogen responsible for this proliferation has been localized to the axon surface and biochemically characterized, it has yet to be identified. Recently a family of proteins known as heregulins (HRGs) has been isolated, characterized, and shown to interact with a number of class 1 receptor tyrosine kinases, including the erbB2, erbB3, and erbB4 gene products. These factors include glial growth factor, a Schwann cell mitogen. We have tested the effects of antibodies against components of this system (HRG beta 1 and p185erbB2) in cocultures of rat sensory neurons and human (or rat) Schwann cells to elucidate the role of these proteins in axon-induced Schwann cell proliferation. 2C4, a monoclonal antibody specific for the human p185erbB2 receptor tyrosine kinase, bound to the surface of human Schwann cells and reduced human Schwann cell incorporation of [3H]thymidine by > 90% compared with untreated controls in this coculture system. This antibody had no effect on rat Schwann cell incorporation of [3H]thymidine under similar conditions. A polyclonal antibody raised against HRG beta 1 reduced human and rat Schwann cell incorporation of [3H]thymidine by nearly 80% and up to 49%, respectively, relative to controls. These results imply that a HRG, or a HRG-like molecule, is a component of the axonal mitogen. This mitogen is presented to Schwann cells by axons and induces proliferation through an interaction that involves p185erbB2 on Schwann cells.
Smooth muscle cell-derived carbon monoxide is a regulator of vascular cGMP.
Authors: Morita T , Perrella M A , Lee M E , Kourembanas S
Published: May 2006

Carbon monoxide (CO) is a product of the enzyme heme oxygenase (HO; EC 1.14.99.3). In vascular smooth muscle cells, exogenously administered CO increases cyclic guanosine 3',5'-monophosphate (cGMP), which is an important regulator of vessel tone. We report here that smooth muscle cells produce CO via HO and that it regulates cGMP levels in these cells. Hypoxia, which has profound effects on vessel tone, significantly increased the transcriptional rate of the HO-1 gene resulting in corresponding increases of its mRNA and HO enzymatic activity. In addition, under the same conditions, rat aortic and pulmonary artery smooth muscle cells accumulated high levels of cGMP following a similar time course to that of HO-1 production. The increased accumulation of cGMP in smooth muscle cells required the enzymatic activity of HO, since it was abolished by a specific HO inhibitor, tin protoporphyrin. In contrast, N omega-nitro-L-arginine, a potent inhibitor of nitric oxide (NO) synthesis, had no effect on cGMP produced by smooth muscle cells, indicating that NO is not responsible for the activation of guanylyl cyclase in this setting. Furthermore, conditioned medium from hypoxic smooth muscle cells stimulated cGMP production in recipient cells and this stimulation was completely inhibited by tin protoporphyrin or hemoglobin, an inhibitor of CO production and a scavenger of CO, respectively. This report shows that HO-1 is expressed by vascular smooth muscle cells and that its product, CO, may regulate vascular tone under physiologic and pathophysiologic (such as hypoxic) conditions.
A second N-acylhomoserine lactone signal produced by Pseudomonas aeruginosa.
Authors: Pearson J P , Passador L , Iglewski B H , Greenberg E P
Published: May 2006

Quorum sensing systems are used by a number of Gram-negative bacterial species to regulate specific sets of genes in a cell density-dependent manner. Quorum sensing involves synthesis and detection of extracellular signals termed autoinducers. As shown in recombinant Escherichia coli, the Pseudomonas aeruginosa autoinducer (PAI) N-(3-oxododecanoyl)homoserine lactone, together with the lasR gene product, activate the P. aeruginosa lasB gene. In this study, PAI was shown to activate lasB-lacZ expression in a P. aeruginosa lasR mutant containing a plasmid with lasR under the control of the lac promoter. The concentration of PAI necessary for half-maximal activation of the lasB-lacZ fusion was approximately 1 microM, which is within the range of PAI levels found in P. aeruginosa culture fluids. The effect of PAI on a P. aeruginosa lasR mutant containing a plasmid with lasR under the control of its own promoter and containing the lasB-lacZ fusion was also tested. Although extracts of culture fluid activated the lasB promoter in this construct, concentrations of PAI as high as 10 microM did not. This indicates the presence of a second extracellular factor (factor 2) that is required for lasB activation in P. aeruginosa when lasR is controlled by its own promoter but not when lasR is controlled by a strong foreign promoter. Factor 2 was shown to be N-butyrylhomoserine lactone. Although recombinant E. coli cells containing the PAI synthase gene, lasI, produce PAI, these cells do not produce factor 2. Furthermore, a P. aeruginosa mutant that produced about 0.1% of the wild-type level of PAI made about 5% of the wild-type level of factor 2. This indicates that factor 2 synthesis results from the activity of a gene product other than PAI synthase. The role of factor 2 in virulence gene regulation remains to be determined, but this compound may affect the expression of lasR, which in turn activates transcription of numerous virulence genes in the presence of sufficient PAI. Apparently, multiple quorum sensing systems can occur and interact with each other in a single bacterial species.
Regulation of leukocyte-endothelium interaction and leukocyte transendothelial migration by intercellular adhesion molecule 1-fibrinogen recognition.
Authors: Languino L R , Duperray A , Joganic K J , Fornaro M , Thornton G B , Altieri D C
Published: May 2006

Although primarily recognized for its role in hemostasis, fibrinogen is also required for competent inflammatory reactions in vivo. It is now shown that fibrinogen promotes adhesion to and migration across an endothelial monolayer of terminally differentiated myelomonocytic cells. This process does not require chemotactic/haptotactic gradients or cytokine stimulation of the endothelium and is specific for the association of fibrinogen with intercellular adhesion molecule 1 (ICAM-1) on endothelium. Among other adhesive plasma proteins, fibronectin fails to increase the binding of leukocytes to endothelium, or transendothelial migration, whereas vitronectin promotes the binding but not the migration. The fibrinogen-mediated leukocyte adhesion and transendothelial migration could be inhibited by a peptide from the fibrinogen gamma-chain sequence N117NQKIVNL-KEKVAQLEA133, which blocks the binding of fibrinogen to ICAM-1. This interaction could also be inhibited by new anti-ICAM-1 monoclonal antibodies that did not affect the ICAM-1-CD11a/CD18 recognition, thus suggesting that the fibrinogen binding site on ICAM-1 may be structurally distinct from regions previously implicated in leukocyte-endothelium interaction. Therefore, binding of fibrinogen to vascular cell receptors is sufficient to initiate (i) increased leukocyte adhesion to endothelium and (ii) leukocyte transendothelial migration. These two processes are the earliest events of immune inflammatory responses and may also contribute to atherosclerosis.
Recombinant rat CBF-C, the third subunit of CBF/NFY, allows formation of a protein-DNA complex with CBF-A and CBF-B and with yeast HAP2 and HAP3.
Authors: Sinha S , Maity S N , Lu J , de Crombrugghe B
Published: May 2006

The CCAAT binding factor CBF is a heteromeric transcription factor, which binds to functional CCAAT motifs in many eukaryotic promoters. cDNAs for the A and B subunits of CBF (CBF-A and CBF-B) and for their yeast homologues HAP3 and HAP2 have been previously isolated, but the purified recombinant CBF-A and CBF-B together are unable to bind to CCAAT motifs in DNA. Here we report the isolation of a cDNA coding for rat CBF-C, demonstrate that recombinant CBF-C is required together with CBF-A and CBF-B to form a CBF-DNA complex, and show that CBF-C is present in this protein-DNA complex together with the other two subunits. We further show that CBF-C allows formation of a complex between the purified recombinant yeast HAP2 and HAP3 polypeptides and a CCAAT-containing DNA and is present in this complex, implying the existence of a CBF-C homologue in yeast. We show that CBF-A and CBF-C interact with each other to form a CBF-A-CBF-C complex and that CBF-B does not interact with CBF-A or CBF-C individually but that it associates with the CBF-A-CBF-C complex. Our results indicate that CBF is a unique evolutionarily conserved DNA binding protein.
The p150Glued component of the dynactin complex binds to both microtubules and the actin-related protein centractin (Arp-1).
Authors: Waterman-Storer C M , Karki S , Holzbaur E L
Published: May 2006

p150Glued was first identified as a polypeptide that copurifies with cytoplasmic dynein, the minus-end-directed microtubule-based motor protein, and has more recently been shown to be present as a member of the oligomeric dynactin complex, which includes the actin-related protein centractin (Arp-1). Dynactin is thought to mediate dynein-driven vesicle motility, as well as nuclear transport, in lower eukaryotes. The mechanism by which dynactin may function in these cellular processes is unknown. To examine the role of the dynactin complex in vivo, we overexpressed the rat cDNA encoding p150Glued in Rat-2 fibroblasts. Overexpression of full-length, as well as C-terminal deletion, constructs resulted in the decoration of microtubules with the p150Glued polypeptides. This cellular evidence for microtubule association was corroborated by in vitro microtubule-binding assays. Amino acids 39-150 of p150Glued were determined to be sufficient for microtubule association. We also tested for a direct interaction between p150Glued and centractin. In vitro translated centractin was specifically retained by a p150Glued affinity column, and this interaction was blocked by a synthetic peptide which corresponds to a highly conserved motif from the C terminus of p150Glued. These results demonstrate that p150Glued, a protein implicated in cytoplasmic dynein-based microtubule motility, is capable of direct binding to both microtubules and centractin.
Soluble type II interleukin 1 (IL-1) receptor binds and blocks processing of IL-1 beta precursor and loses affinity for IL-1 receptor antagonist.
Authors: Symons J A , Young P R , Duff G W
Published: May 2006

Two IL-1 receptors have been identified, termed type I and type II. The extracellular domain of the type II IL-1 receptor is released from certain cells and can function as a specific inhibitor of IL-1 beta activity. We assessed the ligand-binding properties of the type II membrane-bound and soluble IL-1 receptor (sIL-1R) from the human B cell line Raji by competition. Upon release, the affinity of sIL-1R for IL-1 alpha and IL-1 beta remained constant, and both soluble and cell surface IL-1 receptors bound to the same regions on the IL-1 beta molecule as defined by binding of a series of IL-1 beta mutant molecules. However, the affinity of sIL-1R for the IL-1 receptor antagonist (IL-1ra) decreased by a factor of 2000 when compared with the cell surface receptor. Type II sIL-1R and IL-1ra had an additive effect in inhibiting the binding of IL-1 beta to cell surface IL-1 receptors. In contrast, the combination of recombinant type 1 sIL-1R with IL-1ra abrogated the inhibition seen with each of the individual agents alone. The type II cell surface IL-1 receptor failed to bind the biologically inactive IL-1 beta precursor molecule, but binding to the IL-1 beta precursor was observed on cellular release of the receptor; this was confirmed with 35S-labeled IL-1 beta. Binding of IL-1 beta precursor by sIL-1R inhibited the precursor's ability to be processed to the mature, biologically active 17-kDa species. These observations suggest that the type II sIL-1R inhibits IL-1 beta at two steps, by preventing processing of propeptide and by blocking the interaction of mature IL-1 beta with type I IL-1 receptor. In addition, type II sIL-1R does not interfere with inhibition mediated by IL-1ra.
Site-directed mutagenesis studies of the high-affinity streptavidin-biotin complex: contributions of tryptophan residues 79, 108, and 120.
Authors: Chilkoti A , Tan P H , Stayton P S
Published: May 2006

We report the functional characterization of site-directed biotin binding-site mutants of recombinant core streptavidin. The mutagenesis studies were aimed at characterizing the contributions of Trp residues known to contact biotin that have been postulated to control the exceptional binding affinity observed in this system. The functional properties of single site-directed mutants replacing Trp residues with Phe or Ala at positions 79, 108, and 120 were investigated by quantitating the EC50 binding parameters of these mutants to biotin and 2-iminobiotin in an ELISA format. The biotin EC50 for all mutants was the same as wild-type streptavidin, demonstrating that their delta Ka values relative to wild type were < 10(6). The conservative W79F and W108F mutants displayed only a 2- to 3-fold increase in EC50 for 2-iminobiotin, corresponding to an estimated delta Ka < 10, while the W120F mutant displayed a much greater alteration in 2-iminobiotin EC50, corresponding to an estimated delta Ka of 10(2). These delta Ka values are likely to reflect similar changes for biotin. The 2-iminobiotin EC50 values for the Ala mutants fell outside the accessible concentration range of the ELISA assay, demonstrating that these mutations lowered the Ka by a factor of 10(4) to 10(6). Direct estimation of biotin Ka values for W79A, W120A, and W120F in an ultrafiltration binding assay yielded Ka values of 4.3 x 10(7) M-1, 8.6 x 10(6) M-1, and > 5 x 10(9) M-1, respectively, in excellent agreement with the ELISA estimates of delta Ka with 2-iminobiotin as a reporter ligand. The results of these preliminary functional studies suggest that these aromatic side chains contribute significantly to the streptavidin-biotin binding free energy.
Taste receptor cells arise from local epithelium, not neurogenic ectoderm.
Authors: Stone L M , Finger T E , Tam P P , Tan S S
Published: May 2006

Except for taste bud cells, all sensory receptor cells and neurons have been shown to originate from neurogenic ectoderm (i.e., neural tube, neural crest, or ectodermal placodes). Descriptive studies on taste buds indicate that they, however, may arise from local epithelium. To determine whether taste receptor cells originate from neurogenic ectoderm or from local epithelium, the tongues of X chromosome-inactivation mosaic mice were examined. Results of this analysis show that taste bud cells and their surrounding epithelium always match in terms of the mosaic marker. This suggests that taste cells and epithelial cells arise from a common progenitor and that taste receptor cells originate from local tissue elements. Since taste buds are widespread in the oropharynx, they lie in epithelium derived from both ectoderm and endoderm. Therefore, taste receptor cells can be induced in tissue from two different germ layers. Thus in terms of tissues of origin, taste receptor cells are unlike other cells with neuronal characteristics.
Amphibian development in the virtual absence of gravity.
Authors: Souza K A , Black S D , Wassersug R J
Published: May 2006

To test whether gravity is required for normal amphibian development, Xenopus laevis females were induced to ovulate aboard the orbiting Space Shuttle. Eggs were fertilized in vitro, and although early embryonic stages showed some abnormalities, the embryos were able to regulate and produce nearly normal larvae. These results demonstrate that a vertebrate can ovulate in the virtual absence of gravity and that the eggs can develop to a free-living stage.