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Showing papers by "Rockefeller University published in 1990"


Journal ArticleDOI
21 Sep 1990-Science
TL;DR: CD14, a differentiation antigen of monocytes, was found to bind complexes of LPS and LBP, and blockade of CD14 with monoclonal antibodies prevented synthesis of TNF-alpha by whole blood incubated with LPS.
Abstract: Leukocytes respond to lipopolysaccharide (LPS) at nanogram per milliliter concentrations with secretion of cytokines such as tumor necrosis factor-alpha (TNF-alpha). Excess secretion of TNF-alpha causes endotoxic shock, an often fatal complication of infection. LPS in the bloodstream rapidly binds to the serum protein, lipopolysaccharide binding protein (LBP), and cellular responses to physiological levels of LPS are dependent on LBP. CD14, a differentiation antigen of monocytes, was found to bind complexes of LPS and LBP, and blockade of CD14 with monoclonal antibodies prevented synthesis of TNF-alpha by whole blood incubated with LPS. Thus, LPS may induce responses by interacting with a soluble binding protein in serum that then binds the cell surface protein CD14.

4,048 citations


Journal ArticleDOI
21 Sep 1990-Science
TL;DR: The identification of this pathway for LPS-induced monocyte stimulation may aid in the development of treatments for diseases in which Gram-negative sepsis or endotoxemia are involved.
Abstract: The primary structure of lipopolysaccharide binding protein (LBP), a trace plasma protein that binds to the lipid A moiety of bacterial lipopolysaccharides (LPSs), was deduced by sequencing cloned complementary DNA. LBP shares sequence identity with another LPS binding protein found in granulocytes, bactericidal/permeability-increasing protein, and with cholesterol ester transport protein of the plasma. LBP may control the response to LPS under physiologic conditions by forming high-affinity complexes with LPS that bind to monocytes and macrophages, which then secrete tumor necrosis factor. The identification of this pathway for LPS-induced monocyte stimulation may aid in the development of treatments for diseases in which Gram-negative sepsis or endotoxemia are involved.

1,671 citations


Journal ArticleDOI
TL;DR: It is demonstrated that gonadal steroids are necessary for the maintenance of normal adult CA1 hippocampal pyramidal cell structure and implies that CA1 pyramsidal cell dendritic spine density may fluctuate during the normal rat estrous cycle.
Abstract: Gonadal steroids are known to influence hippocampal physiology in adulthood. It is presently unknown whether gonadal steroids influence the morphology of hippocampal neurons in the adult intact rat brain. In order to determine whether female sex hormones influence hippocampal morphology in the intact adult, we performed Golgi impregnation on brains from ovariectomized rats and ovariectomized rats which received estradiol or estradiol and progesterone replacement. Removal of circulating gonadal steroids by ovariectomy of adult female rats resulted in a profound decrease in dendritic spine density in CA1 pyramidal cells of the hippocampus. Estradiol replacement prevented the observed decrease in dendritic spine density; progesterone augmented the effect of estradiol within a short time period (5 hr). Ovariectomy or gonadal steroid replacement did not affect spine density of CA3 pyramidal cells or granule cells of the dentate gyrus. These results demonstrate that gonadal steroids are necessary for the maintenance of normal adult CA1 hippocampal pyramidal cell structure. The short time course required to observe these effects (3 d for the estradiol effect and 5 hr for the progesterone effect) implies that CA1 pyramidal cell dendritic spine density may fluctuate during the normal (4-5 d) rat estrous cycle.

1,353 citations


Journal ArticleDOI
TL;DR: These results demonstrate rapid and ongoing dendritic plasticity in a specific population of hippocampal neurons in experimentally unmanipulated animals.
Abstract: We have used Golgi-impregnated tissue to demonstrate that apical dendritic spine density in CA1 hippocampal pyramidal cells undergoes a cyclic fluctuation as estradiol and progesterone levels vary across the estrous cycle in the adult female rat We observed a 30% decrease in apical dendritic spine density over the 24-hr period between the late proestrus and the late estrus phases of the cycle Spine density then appears to cycle back to proestrus values over a period of several days In contrast, no significant changes in dendritic spine density across the estrous cycle occur in CA3 pyramidal cells or dentate gyrus granule cells These results demonstrate rapid and ongoing dendritic plasticity in a specific population of hippocampal neurons in experimentally unmanipulated animals

1,040 citations


Journal ArticleDOI
TL;DR: The changes in dendritic morphology observed may be indicative of neurons in the early stages of degeneration, as prolonged exposure to high levels of corticosterone has been shown by others to result in a loss of CA3 pyramidal cells.

1,010 citations


Journal ArticleDOI
TL;DR: DNA-binding and antisera reactivity data suggest that HNF-4 could be identical to liver factor A1 (LF-A1), a DNA-binding activity implicated in the regulation of transcription of the alpha 1-antitrypsin, apolipoprotein A1, and pyruvate kinase genes.
Abstract: HNF-4 (hepatocyte nuclear factor 4) is a protein enriched in liver extracts that binds to sites required for the transcription of the genes for transthyretin (TTR), the carrier protein in the serum for vitamin A and thyroid hormone, and for apolipoprotein CIII (apoCIII), a major constituent of chylomicrons and very low-density lipoproteins (VLDL) Synthetic oligonucleotides derived from amino acid sequence of affinity-purified HNF-4 protein (54 kD) were used in the polymerase chain reaction (PCR) to isolate a cDNA clone encoding the protein HNF-4 is a member of the steroid hormone receptor superfamily with an unusual amino acid in the conserved "knuckle" of the first zinc finger (DGCKG) Studies with in vitro-translated HNF-4 protein show that it binds to its recognition site as a dimer, and cotransfection assays indicate that it activates transcription in a sequence-specific fashion in nonhepatic (HeLa) cells Northern blot analysis reveals that HNF-4 mRNA is present in kidney and intestine, as well as liver, but is absent in other tissues DNA-binding and antisera reactivity data suggest that HNF-4 could be identical to liver factor A1 (LF-A1), a DNA-binding activity implicated in the regulation of transcription of the alpha 1-antitrypsin, apolipoprotein A1, and pyruvate kinase genes The similarity between HNF-4 and other ligand-dependent transcription factors raises the possibility that HNF-4 and the genes it regulates respond to an as yet unidentified ligand

1,002 citations


Journal ArticleDOI
09 Mar 1990-Science
TL;DR: An antibody to a platelet integral membrane glycoprotein was found to cross-react with the previously identified CD31 myelomonocytic differentiation antigen and with hec7, an endothelial cell protein that is enriched at intercellular junctions.
Abstract: An antibody to a platelet integral membrane glycoprotein was found to cross-react with the previously identified CD31 myelomonocytic differentiation antigen and with hec7, an endothelial cell protein that is enriched at intercellular junctions. This antibody identified a complementary DNA clone from an endothelial cell library. The 130-kilodalton translated sequence contained six extracellular immunoglobulin (Ig)-like domains and was most similar to the cell adhesion molecule (CAM) subgroup of the Ig superfamily. This is the only known member of the CAM family on platelets. Its cell surface distribution suggests participation in cellular recognition events.

973 citations


Journal ArticleDOI
TL;DR: It is concluded that increased TNF production is a normal host response to P falciparum infection, but that excessive levels of production may predispose to cerebral malaria and a fatal outcome.

861 citations


Journal ArticleDOI
TL;DR: The geographic distribution of pneumococci resistant to one or more of the antibiotics penicillin, erythromycin, trimethoprim-sulfamethoxazole, and tetracycline appears to be expanding, and there exist foci of resistance to chloramphenicol and rifampin.
Abstract: The geographic distribution of pneumococci resistant to one or more of the antibiotics penicillin, erythromycin, trimethoprim-sulfamethoxazole, and tetracycline appears to be expanding, and there exist foci of resistance to chloramphenicol and rifampin. Multiply resistant pneumococci are being encountered more commonly and are more often community acquired. Factors associated with infection caused by resistant pneumococci include young age, duration of hospitalization, infection with a pneumococcus of serogroup 6, 19, or 23 or serotype 14, and exposure to antibiotics to which the strain is resistant. At present, the most useful drugs for the management of resistant pneumococcal infections are cefotaxime, ceftriaxone, vancomycin, and rifampin. If the strains are susceptible, chloramphenicol may be useful as an alternative, less expensive agent. Appropriate interventions for the control of resistant pneumococcal outbreaks include investigation of the prevalence of resistant strains, isolation of patients, possible treatment of carriers, and reduction of usage of antibiotics to which the strain is resistant. The molecular mechanisms of penicillin resistance are related to the structure and function of penicillin-binding proteins, and the mechanisms of resistance to other agents involved in multiple resistance are being elucidated. Recognition is increasing of the standard screening procedure for penicillin resistance, using a 1-microgram oxacillin disk.

844 citations


Journal ArticleDOI
TL;DR: The anatomical and functional organization of the inferior parietal lobule was investigated in macaque monkeys by using anterograde and retrograde anatomical tracing techniques and single cell recording techniques in awake, behaving monkeys.
Abstract: The anatomical and functional organization of the inferior parietal lobule was investigated in macaque monkeys by using anterograde and retrograde anatomical tracing techniques and single cell recording techniques in awake, behaving monkeys. The connections of areas 7a and 7b, and of two previously unexplored areas, the lateral intraparietal area (LIP) and the dorsal prelunate area (DP), were examined in detail. Functional mapping experiments were performed in all four areas. Prior to this study the pathways for visual input to area 7a were unclear. In these experiments we found several direct projections from extrastriate visual areas, including the lateral intraparietal (LIP), dorsal prelunate (DP), parieto-occipital (PO), and medial superior temporal (MST) areas into area 7a. Using the observed laminar patterns of connections between areas 7a, LIP, and DP and other extrastriate cortical areas, we were able to construct a hypothetical flow of visual information processing from striate cortex to area 7a. A broader hierarchy was also produced, which relates the positions of areas 7a, 7b, LIP, and DP to various cortical fields in the parietal, temporal, and frontal lobes. By combining single cell recording techniques in trained monkeys with anatomical tracing techniques, we have parceled the inferior parietal lobule into several subdivisions on the basis of both anatomical and physiological grounds. A clear segregation of visual and somatosensory responses was found in the inferior parietal lobule with areas 7a, LIP, and DP being visual and visual-motor and area 7b being primarily somatosensory. A similar segregation was found anatomically with areas 7a, LIP, and DP being interconnected primarily with other visual cortical areas and area 7b being connected with several somatosensory areas. Area 7b was also found to connect to a few visual cortical areas, and these connections likely account for the small but consistent number of visually responsive cells that are found in this region. Areas LIP, DP, and 7a differed in receptive field and saccade-related properties. Area 7a visual receptive fields were very large and usually bilateral with a small but significant number of them having receptive field centers in the ipsilateral visual field. Area DP and LIP receptive fields were smaller and the receptive field peaks were almost always confined to the contralateral visual field. Areas 7a, DP, and LIP all contained cells with saccade-related responses; however, in area 7a there were fewer saccade cells than area LIP, and presaccadic responses were only observed in area LIP.(ABSTRACT TRUNCATED AT 400 WORDS)

823 citations


Journal ArticleDOI
27 Jul 1990-Science
TL;DR: A high spatial resolution optical imaging system was developed to visualize cerebral cortical activity in vivo and found no ocular dominance organization was seen, while regions of poor orientation tuning colocalized to every other cytochrome oxidase stripe.
Abstract: A high spatial resolution optical imaging system was developed to visualize cerebral cortical activity in vivo. This method is based on activity-dependent intrinsic signals and does not use voltage-sensitive dyes. Images of the living monkey striate (VI) and extrastriate (V2) visual cortex, taken during visual stimulation, were analyzed to yield maps of the distribution of cells with various functional properties. The cytochrome oxidase--rich blobs of V1 and the stripes of V2 were imaged in the living brain. In V2, no ocular dominance organization was seen, while regions of poor orientation tuning colocalized to every other cytochrome oxidase stripe. The orientation tuning of other regions of V2 appeared organized as modules that are larger and more uniform than those in V1.

Journal ArticleDOI
16 Nov 1990-Science
TL;DR: The expression patterns conferred by specific combinations of 35S subdomains differ in tobacco and petunia, indicating that a combinatorial code of cisregulatory elements may be interpreted differently in different species.
Abstract: Appropriate regulation of transcription in higher plants requires specific cis elements in the regulatory regions of genes and their corresponding trans-acting proteins. Analysis of the cauliflower mosaic virus (CaMV) 35S promoter has contributed to the understanding of transcriptional regulatory mechanisms. The intact 35S promoter confers constitutive expression upon heterologous genes in most plants. Dissection into subdomains that are able to confer tissue-specific gene expression has demonstrated that the promoter has a modular organization. When selected subdomains are combined, they confer expression not detected from the isolated subdomains, suggesting that synergistic interactions occur among cis elements. The expression patterns conferred by specific combinations of 35S subdomains differ in tobacco and petunia. This indicates that a combinatorial code of cisregulatory elements may be interpreted differently in different species.

Journal ArticleDOI
07 Sep 1990-Cell
TL;DR: The DNA binding subunit of the transcription factor NF-kappa B, p50, has been cloned and sequence analysis reveals remarkable homology for over 300 amino acids at the amino-terminal end to the oncogene v-rel, its cellular homolog c-rel and the Drosophila maternal effect gene dorsal as mentioned in this paper.

Journal ArticleDOI
TL;DR: It is shown that mouse T cells can be reliably primed in situ using dendritic cells as APC and a small but relevant vacuolar system for presenting antigens over a several day period in situ is suggested.
Abstract: T cells recognize peptides that are bound to MHC molecules on the surface of different types of antigen-presenting cells (APC). Antigen presentation most often is studied using T cells that have undergone priming in situ, or cell lines that have been chronically stimulated in vitro. The use of primed cells provides sufficient numbers of antigen-reactive lymphocytes for experimental study. A more complete understanding of immunogenicity, however, requires that one develop systems for studying the onset of a T cell response from unprimed lymphocytes, especially in situ. Here it is shown that mouse T cells can be reliably primed in situ using dendritic cells as APC. The dendritic cells were isolated from spleen, pulsed with protein antigens, and then administered to naive mice. Antigen-responsive T cells developed in the draining lymphoid tissue, and these T cells only recognized protein when presented on cells bearing the same MHC products as the original priming dendritic cells. In contrast, little or no priming was seen if antigen-pulsed spleen cells or peritoneal cells were injected. Since very small amounts of the foreign protein were visualized within endocytic vacuoles of antigen-pulsed dendritic cells, it is suggested that dendritic cells have a small but relevant vacuolar system for presenting antigens over a several day period in situ.

Journal ArticleDOI
TL;DR: The D enantiomers of three naturally occurring antibiotics--cecropin A, magainin 2 amide, and melittin--were synthesized and it is suggested that the mode of action of these peptides on the membranes of bacteria, erythrocytes, plasmodia, and artificial lipid bilayers may be similar and involves the formation of ion-channel pores spanning the membranes, but without specific interaction with chiral receptors or enzymes.
Abstract: The D enantiomers of three naturally occurring antibiotics--cecropin A, magainin 2 amide, and melittin--were synthesized. In addition, the D enantiomers of two synthetic chimeric cecropin-melittin hybrid peptides were prepared. Each D isomer was shown by circular dichroism to be a mirror image of the corresponding L isomer in several solvent mixtures. In 20% hexafluoro-2-propanol the peptides contained 43-75% alpha-helix. The all-D peptides were resistant to enzymatic degradation. The peptides produced single-channel conductances in planar lipid bilayers, and the D and L enantiomers caused equivalent amounts of electrical conductivity. All of the peptides were potent antibacterial agents against representative Gram-negative and Gram-positive species. The D and L enantiomers of each peptide pair were equally active, within experimental error. Sheep erythrocytes were lysed by both D- and L-melittin but not by either isomer of cecropin A, magainin 2 amide, or the hybrids cecropin A-(1-13)-melittin-(1-13)-NH2 or cecropin A-(1-8)-melittin-(1-18)-NH2. The infectivity of the bloodstream form of the malaria parasite Plasmodium falciparum was also inhibited by the D and L hybrids. It is suggested that the mode of action of these peptides on the membranes of bacteria, erythrocytes, plasmodia, and artificial lipid bilayers may be similar and involves the formation of ion-channel pores spanning the membranes, but without specific interaction with chiral receptors or enzymes.

Journal ArticleDOI
TL;DR: The p150, 90 heterodimer is the major beta 2 integrin of spleen dendritic cells, and it is speculated that it may function to localize these APC at sites that permit access to the recirculating pool of resting T cells.
Abstract: Hybridoma fusions with hamster hosts were undertaken to generate mAbs to mouse spleen dendritic cells. Two mAb were obtained and used to uncover the distinct integrins of these APC. One, 2E6, bound a determinant common to all members of the CD11/CD18 family, most likely the shared 90 kD CD18 beta chain. 2E6 immunoprecipitated the characteristic beta 2 integrin heterodimers from lymphocytes (p180, 90; CD11a) and macrophages (p170,90; CD11b), but from dendritic cells, a p150,90 (presumably CD11c) integrin was the predominant species. 2E6 inhibited the binding function of the CD11a and CD11b integrins on B cells and macrophages in appropriate assays, but 2E6 exerted little or no inhibition on the clustering of dendritic cells to T cells early in primary MLR, suggesting a CD11/CD18-independent mechanism for this binding. The second mAb, N418, precipitated a 150, 90 kD heterodimer that shared the 2E6 CD18 epitope. This N418 epitope may be the murine homologue of the previously characterized human CD11c molecule, but the epitope was only detected on dendritic cells. N418 did not react with peritoneal macrophages, anti-Ig-induced spleen B blasts, or bulk lymph node cells. When used to stain sections of spleen, N418 stained dendritic cells in the T-dependent areas, much like anti-class II mAbs that were also generated in these fusions. In addition, N418 revealed nests of dendritic cells that punctuated the rim of marginal zone macrophages between red and white pulp. This localization positioned most dendritic cells at regions where arterial vessels and T cells enter the white pulp. We conclude that the p150, 90 heterodimer is the major beta 2 integrin of spleen dendritic cells, and we speculate that it may function to localize these APC at sites that permit access to the recirculating pool of resting T cells.

Journal ArticleDOI
TL;DR: The results show that the filter characteristics of striate cortical cells are not necessarily fixed, but can be dynamic, changing according to context, and this study modeled a neuronal ensemble encoding orientation.


Journal ArticleDOI
17 Aug 1990-Science
TL;DR: Overexpression of apolipoprotein CIII can be a primary cause of hypertriglyceridemia in vivo and may provide one possible etiology for this common disorder in humans.
Abstract: Primary and secondary hypertriglyceridemia is common in the general population, but the biochemical basis for this disease is largely unknown. With the use of transgenic technology, two lines of mice were created that express the human apolipoprotein CIII gene. One of these mouse lines with 100 copies of the gene was found to express large amounts of the protein and to be severely hypertriglyceridemic. The other mouse line with one to two copies of the gene expressed low amounts of the protein, but nevertheless manifested mild hypertriglyceridemia. Thus, overexpression of apolipoprotein CIII can be a primary cause of hypertriglyceridemia in vivo and may provide one possible etiology for this common disorder in humans.

Journal ArticleDOI
19 Oct 1990-Cell
TL;DR: A cloverleaf structure in poliovirus RNA plays a central role in organizing viral and cellular proteins involved in positive strand production.

Journal ArticleDOI
TL;DR: The structure of different cDNA clones indicates that USF RNA is differentially spliced, and alternative exon usage may regulate the levels of functional USF protein.
Abstract: We isolated full-length cDNAs encoding the 43-kD form of human upstream stimulatory factor (USF), a cellular factor required for efficient transcription of the adenovirus major late (AdML) promoter in vitro. Sequence analysis showed USF to be a member of the c-myc-related family of DNA-binding proteins. Using proteins translated in vitro, we identified a DNA-binding domain near the carboxyl terminus, which includes both a helix-loop-helix motif and a leucine repeat. We show that USF interacts with its target DNA as a dimer. The leucine repeat is required for efficient DNA binding of the intact protein and for interactions between full-length and truncated USF proteins. Interestingly, it is not required for DNA binding of the isolated helix-loop-helix domain. The structure of different cDNA clones indicates that USF RNA is differentially spliced, and alternative exon usage may regulate the levels of functional USF protein.

Journal ArticleDOI
TL;DR: Serological Cross-Reactions and Indications of a Glycolipid Anchor and the Hydrophobic Constituents are studied.
Abstract: CHARACTERIZATION OF GLYCOLIPID ANCHORS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 Indications of a Glycolipid Anchor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . 5 Chemical Analysis . 5 The Ethanolamine-Glycan-Inositol Core . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 Variations on the Core Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 The Hydrophobic Constituents. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 Serological Cross-Reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 1

Journal ArticleDOI
TL;DR: Experiments presented here demonstrate that PMN exposed to NAP-1/IL-8 expressed increased amounts of CD11b/CD18, as well as CD11c/ CD18 and CR1, on their cell surface, while expression of Fc gamma RIII and HLA-A,B,C remained essentially unchanged.
Abstract: The cytokine NAP-1/IL-8 is produced by a variety of different cells in response to inflammatory stimuli and elicits several biological responses from PMN. Experiments presented here demonstrate that PMN exposed to NAP-1/IL-8 expressed increased amounts of CD11b/CD18, as well as CD11c/CD18 and CR1, on their cell surface, while expression of Fc gamma RIII and HLA-A,B,C remained essentially unchanged. Increased CD11b/CD18 and CD11c/CD18 appears to correspond with the release of specific granules by NAP-1/IL-8. NAP-1/IL-8 was also a potent stimulator of several of the binding activities of CD11b/CD18. Ligation of EC3bi by CD11b/CD18 was rapidly enhanced by NAP-1/IL-8, but phagocytosis of the ligated particles was not induced by the agonist. In addition, enhanced binding of EC3bi was observed in the absence of an increase in receptor expression as shown with PMN cytoplasts. NAP-1/IL-8 promoted additional adhesive interactions between CD11b/CD18 and the biosynthetic precursor of LPS, lipid IVa, fibrinogen, and endothelial cells, suggesting that NAP-1/IL-8 may promote leukocyte adhesion in vivo that could lead to recruitment of PMN to sites of tissue inflammation.

Journal ArticleDOI
TL;DR: The observation that different light responses are mediated through distinct photoreceptors raises the question of whether genes that respond to more than one wavelength do so through distinct c/s-acting elements or whether the signal transduction pathways converge to act upon the same regulatory sequence.
Abstract: Light is essential for normal plant growth and development not only as a source of energy but also as a stimulus that regulates numerous developmental and metabolic proc? esses. The plant's responses are varied and complex and dependent upon the quality and quantity of ambient light. The initial requirement for light is as a signal for germination in many plant species. After germination in complete dark? ness, seedlings have a morphology distinct from lightgrown ones and do not express light-inducible genes. Upon illumination of these etiolated seedlings, modifications in the transcription of light-responsive genes occur and rapid light-induced morphological changes ensue. Adaptation of plants in continuous darkness for 2 to 3 days does not cause dramatic morphological changes but results in alter? ations of specific transcript levels (for recent reviews, see Ellis, 1986; Kendrick and Kronenberg, 1986; Cuozzo et al., 1987; Kuhlemeier etal., 1987b; Silverthorne and Tobin, 1987; Jenkins, 1988; Nagy et al., 1988). The most extensively studied light-responsive genes are those encoding the small subunit of ribulose-1,5-bisphos? phate carboxylase-oxygenase (rbcS) and the chlorophyll a/?>-binding proteins (cab) (see Tobin and Silverthorne, 1985; Manzara and Gruissem, 1988; Dean et al., 1989c). In several plant species an increase in the transcript levels from these genes occurs in etiolated seedlings and darkadapted plants in response to light. This increase is me? diated by the photoreceptor phytochrome and is regulated at the transcriptional level (Gallagher and Ellis, 1982; Sil? verthorne and Tobin, 1984; Berry-Lowe and Meagher, 1985; Mosinger et al., 1985). Phytochrome is the best characterized of the three known photoreceptors. The other two, cryptochrome and the UV-B photoreceptor, mediate their effects in response to blue and UV light, respectively (see Kendrick and Kronenberg, 1986). The expression of many light-responsive genes is modulated by more than one wavelength of light (see Tobin and Silverthorne, 1984; Ellis, 1986; Kuhlemeier et al., 1987b). The observation that different light responses are mediated through distinct photoreceptors raises the question of whether genes that respond to more than one wavelength do so through distinct c/s-acting elements or whether the signal transduction pathways converge to act upon the same regulatory sequence. Analyses of the kinetics of light-responsive gene induc? tion show that the rate of mRNA accumulation is variable among genes and can be dependent on the developmental state of the plant (Gallagher et al., 1985; Fluhr and Chua, 1986). This variation may be due to a requirement for distinct regulatory factors or because the genes have different thresholds for a specific regulator. Several genes are down-regulated by light, specifically those encoding phytochrome (Lissemore and Quail, 1988; Kay et al., 1989), NADPH-protochlorophyllide reductase (Batschauer and Apel, 1984; Darrah et al., 1990), and asparagine synthetase (Tsai and Coruzzi, 1990). For each of these genes, the photoresponse is mediated by phyto? chrome. The ability of one photoreceptor to mediate op? posite patterns of expression implies that there is a branch point in the signal transduction pathway leading to these different responses. Studies of many light-regulated genes from different species demonstrate that DNA elements responsible for light-responsive expression are located within 5' upstream sequences (see Kuhlemeier et al., 1987b; Silverthorne and Tobin, 1987; Jenkins, 1988; Benfey and Chua, 1989; Dean et al., 1989a; Stockhaus et al., 1989). However, there is evidence that other regions of the gene can mediate changes in transcript abundance in response to light. For example, in the case of a pea gene encoding ferredoxin, sequences within the transcribed region modulate mRNA levels by affecting transcript stability (Elliot et al., 1989a). In addition, nuclear run-on experiments with petunia rbcS show that both upstream and downstream sequences play a role in the transcriptional regulation of these genes (Dean et al., 1989b). In contrast, downstream sequences of pea rbcS do not affect steady-state transcript abundance (Kuhlemeier et al., 1988b). These differences may reflect subtle variations in the mechanisms that operate to regu? late rbcS expression in different plant species. 1 Current address: Department of Plant Molecular Biology, Leiden University, Clusius Laboratory, Lassenaarseweg 64, 2333 AL Leiden, The Netherlands. 2 To whom correspondence should be addressed.

Journal ArticleDOI
TL;DR: The hypothesis that abnormal protein phosphorylation may play a role in the development of the cerebral amyloidosis that accompanies Alzheimer disease is supported.
Abstract: The turnover and processing of the Alzheimer beta/A4 amyloid precursor protein (beta APP) has been studied in PC12 cells after treatment with agents that regulate protein phosphorylation. Phorbol 12,13-dibutyrate, an agent that stimulates protein kinase C, decreased the levels of mature beta APP and increased the levels of 15- and 19-kDa peptides. These peptides appeared to be COOH-terminal fragments of beta APP, which arose when phorbol 12,13-dibutyrate increased the rate of proteolytic processing of mature forms of beta APP. Okadaic acid, an inhibitor of protein phosphatases 1 and 2A, also led to decreased levels of mature beta APP and increased levels of the 15- and 19-kDa peptides. H-7, an inhibitor of protein kinase C and of several other protein kinases, apparently decreased the rate of proteolytic processing of mature beta APP. The sizes of the putative COOH-terminal fragments observed after treatment with either phorbol 12,13-dibutyrate or okadaic acid suggest that one or both may contain the entire beta/A4 region of beta APP and thus be amyloidogenic. Our results support the hypothesis that abnormal protein phosphorylation may play a role in the development of the cerebral amyloidosis that accompanies Alzheimer disease.

Journal ArticleDOI
TL;DR: The purification of P. falciparum digestive vacuoles is reported and the characterization of the degradative process therein is characterization, finding unique proteolytic organelles designed specifically to degrade hemoglobin.
Abstract: The malaria parasite Plasmodium falciparum uses host erythrocyte hemoglobin as a major nutrient source. We report the purification of P. falciparum digestive vacuoles and characterization of the degradative process therein. Vacuoles were isolated by a combination of differential centrifugation and density gradient separation. The pure vacuoles were capable of degrading hemoglobin to small fragments with a pH optimum of 5-5.5. Proteolysis in the vacuoles appears to be an ordered process, requiring an aspartic protease to clip intact hemoglobin before other proteolytic activities can function efficiently. The vacuoles do not contain other hydrolases commonly found in lysosomes and therefore appear to be unique proteolytic organelles designed specifically to degrade hemoglobin.


Journal ArticleDOI
E Lai1, V R Prezioso, E Smith, O Litvin, R H Costa, James E. Darnell 
TL;DR: The mRNA for HNF-3A is present in the rat liver but not in brain, kidney, intestine, or spleen, and the basis for this difference is cell-specific regulation of HNF -3A gene transcription.
Abstract: Hepatocyte-specific gene expression requires the interaction of many proteins with multiple binding sites in the regulatory regions. HNF-3 is a site found to be important in the maximal hepatocyte-specific expression of several genes. We find that liver nuclear extracts contain three major binding activities for this site, which we call HNF-3A, HNF-3B, and HNF-3C. Purification from rat liver nuclear extracts of HNF-3A and HNF-3C reveals that each activity corresponds to a distinct polypeptide, as determined by SDS-PAGE. Peptide sequence derived from the most abundant species, HNF-3A, was used for synthesizing probes with which to isolate a cDNA clone of this protein. The encoded protein contains 466 amino acids (48.7 kD) and has binding properties identical to those of the purified protein. A 160-amino-acid region that does not resemble the binding domain of any known transcription factor is essential for DNA binding. The mRNA for HNF-3A is present in the rat liver but not in brain, kidney, intestine, or spleen, and the basis for this difference is cell-specific regulation of HNF-3A gene transcription.

Journal ArticleDOI
TL;DR: The multisubunit structure of ISGF3 most likely reflects its participation in receiving a ligand-dependent signal, translocating to the nucleus, and binding to DNA to activate transcription.
Abstract: Interferon-stimulated gene factor 3 (ISGF3) is the ligand-dependent transcriptional activator that, in response to interferon treatment, is assembled in the cell cytoplasm, is translocated to the nucleus, and binds the consensus DNA site, the interferon-stimulated response element. We have purified ISGF3 and identified its constituent proteins: a DNA-binding protein of 48 kDa and three larger polypeptides (84, 91, and 113 kDa), which themselves do not have DNA-binding activity. The multisubunit structure of ISGF3 most likely reflects its participation in receiving a ligand-dependent signal, translocating to the nucleus, and binding to DNA to activate transcription.

Journal ArticleDOI
TL;DR: A protocol that reliably yields preparations that are greater than 80-90% pure is presented, which relies on the sequential depletion of the major cell types in blood and simultaneously provides T cells, monocytes, and B plus natural killer cells for comparison with dendritic cells.
Abstract: Prior studies have identified a subset of dendritic cells in human blood, as well as their stimulatory function for T-cell-mediated immune responses. However research has been limited by difficulties in isolation, since dendritic cells make up only 0.1-1% of blood mononuclear cells. We present a protocol that reliably yields preparations that are greater than 80-90% pure. The method relies on the sequential depletion of the major cell types in blood and simultaneously provides T cells, monocytes, and B plus natural killer cells for comparison with dendritic cells. The last step in the procedure is the removal of residual contaminants on the basis of expression of a CD45R epitope. The enrichment of dendritic cells is evident by three criteria, each of which is related to the surface of these antigen-presenting cells. (i) All dendritic cells are motile, constantly forming large lamellipodia or veils. (ii) When analyzed with a large panel of monoclonal antibodies and the FACS, the cells express high levels of all known polymorphic major histocompatibility complex gene products, as well as a distinct combination of receptors and adhesion molecules. Unlike monocytes, for example, dendritic cells lack Fc receptors and the colony-stimulating factor 1 receptor (c-fms) but express much higher levels of ICAM-1 and LFA-3 adhesins. (iii) In functional assays, dendritic cells are at least 100 times more potent than monocytes or lymphocytes in stimulating the primary mixed leukocyte reaction. These properties help make the trace subset of dendritic cells more amenable to further functional and clinical studies.