Showing papers by "Rockefeller University published in 1994"

Positional cloning of the mouse obese gene and its human homologue

Abstract: The mechanisms that balance food intake and energy expenditure determine who will be obese and who will be lean. One of the molecules that regulates energy balance in the mouse is the obese (ob) gene. Mutation of ob results in profound obesity and type II diabetes as part of a syndrome that resembles morbid obesity in humans. The ob gene product may function as part of a signalling pathway from adipose tissue that acts to regulate the size of the body fat depot.

Jak-STAT pathways and transcriptional activation in response to IFNs and other extracellular signaling proteins

Abstract: Through the study of transcriptional activation in response to interferon alpha (IFN-alpha) and interferon gamma (IFN-gamma), a previously unrecognized direct signal transduction pathway to the nucleus has been uncovered: IFN-receptor interaction at the cell surface leads to the activation of kinases of the Jak family that then phosphorylate substrate proteins called STATs (signal transducers and activators of transcription). The phosphorylated STAT proteins move to the nucleus, bind specific DNA elements, and direct transcription. Recognition of the molecules involved in the IFN-alpha and IFN-gamma pathway has led to discoveries that a number of STAT family members exist and that other polypeptide ligands also use the Jak-STAT molecules in signal transduction.

Production of high titer helper-free retroviruses by transient transfection

Abstract: A method for producing high-titer, helper-free infectious retroviruses is disclosed which employs a novel strategy that uses transient transfection of new retroviral producer cell lines, ecotropic line BOSC 23 and amphotropic line CAK 8, both of which cell lines and their precursor cell lines are disclosed. Because of the advantages over stable packaging cell lines, the BOSC 23 and CAK 8 transient transfection systems greatly facilitate and extend the use of helper-free retroviral vectors. The cell lines and corresponding methods possess wide application in both the medical and biotechnical fields, including gene therapy. These potential applications are disclosed and illustrated.

Long-distance neuronal migration in the adult mammalian brain

Abstract: During the development of the mammalian brain, neuronal precursors migrate to their final destination from their site of birth in the ventricular and subventricular zones (VZ and SVZ, respectively). SVZ cells in the walls of the lateral ventricle continue to proliferate in the brain of adult mice and can generate neurons in vitro, but their fate in vivo is unknown. Here SVZ cells from adult mice that carry a neuronal-specific transgene were grafted into the brain of adult recipients. In addition, the fate of endogenous SVZ cells was examined by microinjection of tritiated thymidine or a vital dye that labeled a discrete population of SVZ cells. Grafted and endogenous SVZ cells in the lateral ventricle of adult mice migrate long distances and differentiate into neurons in the olfactory bulb.

Stat3: a STAT family member activated by tyrosine phosphorylation in response to epidermal growth factor and interleukin-6

Abstract: The STAT family of proteins carries out a dual function: signal transduction and activation of transcription. A new family member, Stat3, becomes activated through phosphorylation on tyrosine as a DNA binding protein in response to epidermal growth factor (EGF) and interleukin-6 (IL-6) but not interferon gamma (IFN-gamma). It is likely that this phosphoprotein forms homodimers as well as heterodimers with the first described member of the STAT family, Stat91 (renamed Stat1 alpha), which is activated by the IFNs and EGF. Differential activation of different STAT proteins in response to different ligands should help to explain specificity in nuclear signaling from the cell surface.

Long-term gene expression and phenotypic correction using adeno-associated virus vectors in the mammalian brain

Abstract: Adeno-associated viral (AAV) vectors are non-pathogenic, integrating DNA vectors in which all viral genes are removed and helper virus is completely eliminated. To evaluate this system in the post-mitotic cells of the brain, we found that an AAV vector containing the lacZ gene (AAVlac) resulted in expression of beta-galactosidase up to three months post-injection in vivo. A second vector expressing human tyrosine hydroxylase (AAVth) was injected into the denervated striatum of unilateral 6-hydroxydopamine-lesioned rats. Tyrosine hydroxylase (TH) immunoreactivity was detectable in striatal neurons and glia for up to four months and we also found significant behavioural recovery in lesioned rats treated with AAVth versus AAVlac controls. Safe and stable TH gene transfer into the denervated striatum may have potential for the genetic therapy of Parkinson's disease.

Characterization of caveolin-rich membrane domains isolated from an endothelial-rich source: implications for human disease.

Abstract: Caveolae are 50-100-nm membrane microdomains that represent a subcompartment of the plasma membrane. Previous morphological studies have implicated caveolae in (a) the transcytosis of macromolecules (including LDL and modified LDLs) across capillary endothelial cells, (b) the uptake of small molecules via a process termed potocytosis involving GPI-linked receptor molecules and an unknown anion transport protein, (c) interactions with the actin-based cytoskeleton, and (d) the compartmentalization of certain signaling molecules, including G-protein coupled receptors. Caveolin, a 22-kD integral membrane protein, is an important structural component of caveolae that was first identified as a major v-Src substrate in Rous sarcoma virus transformed cells. This finding initially suggested a relationship between caveolin, transmembrane signaling, and cellular transformation. We have recently developed a procedure for isolating caveolin-rich membrane domains from cultured cells. To facilitate biochemical manipulations, we have applied this procedure to lung tissue--an endothelial and caveolin-rich source-allowing large scale preparation of these complexes. These membrane domains retain approximately 85% of caveolin and approximately 55% of a GPI-linked marker protein, while they exclude > or = 98% of integral plasma membrane protein markers and > or = 99.6% of other organelle-specific membrane markers tested. Characterization of these complexes by micro-sequencing and immuno-blotting reveals known receptors for modified forms of LDL (scavenger receptors: CD 36 and RAGE), multiple GPI-linked proteins, an anion transporter (plasma membrane porin), cytoskeletal elements, and cytoplasmic signaling molecules--including Src-like kinases, hetero-trimeric G-proteins, and three members of the Rap family of small GTPases (Rap 1--the Ras tumor suppressor protein, Rap 2, and TC21). At least a fraction of the actin in these complexes appeared monomeric (G-actin), suggesting that these domains could represent membrane bound sites for microfilament nucleation/assembly during signaling. Given that the majority of these proteins are known molecules, our current studies provide a systematic basis for evaluating these interactions in vivo.

Lipopolysaccharide (LPS)-binding protein accelerates the binding of LPS to CD14.

Abstract: CD14 is a 55-kD protein found as a glycosylphosphatidylinositol (GPI)-anchored protein on the surface of monocytes, macrophages, and polymorphonuclear leukocytes, and as a soluble protein in the blood. Both forms of CD14 participate in the serum-dependent responses of cells to bacterial lipopolysaccharide (LPS). While CD14 has been described as a receptor for complexes of LPS with LPS-binding protein (LBP), there has been no direct evidence showing whether a ternary complex of LPS, LBP, and CD14 is formed, or whether CD14 binds LPS directly. Using nondenaturing polyacrylamide gel electrophoresis (native PAGE), we show that recombinant soluble CD14 (rsCD14) binds LPS in the absence of LBP or other proteins. Binding of LPS to CD14 is stable and of low stoichiometry (one or two molecules of LPS per rsCD14). Recombinant LBP (rLBP) does not form detectable ternary complexes with rsCD14 and LPS, but it does accelerate the binding of LPS to rsCD14. rLBP facilitates the interaction of LPS with rsCD14 at substoichiometric concentrations, suggesting that LBP functions catalytically, as a lipid transfer protein. Complexes of LPS and rsCD14 formed in the absence of LBP or other serum proteins strongly stimulate integrin function on PMN and expression of E-selectin on endothelial cells, demonstrating that LBP is not necessary for CD14-dependent stimulation of cells. These results suggest that CD14 acts as a soluble and cell surface receptor for LPS, and that LBP may function primarily to accelerate the binding of LPS to CD14.

Human blood contains two subsets of dendritic cells, one immunologically mature and the other immature.

Abstract: Two subsets of dendritic cells, differing in T-cell stimulatory function, have been purified directly from human blood. Both subsets are positive for major histocompatibility complex (MHC) class II expression and negative for lineage-specific antigens (e.g. CD3, CD14, CD16, CD19 negative), but are separated by exploiting differences in expression of the beta 2-integrin, CD11c. The CD11c-negative subset is functionally immature, requiring monocyte-derived cytokines to develop into typical dendritic cells. The CD11c-positive subset has potent T-cell stimulating activity and expresses the activation antigen CD45RO, unlike its immature counterpart. However, these mature cells only develop typical dendritic morphology and high levels of MHC proteins and adhesins after a period of culture independent of exogenous cytokines. Although the freshly isolated mature dendritic cells resemble monocytes in cytospin preparations, the former lack CD14 and have a much stronger primary T-cell stimulatory capacity. We hypothesize that the CD11c-negative immature cells are marrow-derived precursors to tissue dendritic cells, such as epidermal Langerhans' cells, while the CD11c-positive cells are derived from tissues where they have been activated by antigen, and are en route to the spleen or lymph nodes to stimulate T-cell responses there.

Estradiol regulates hippocampal dendritic spine density via an N-methyl-D-aspartate receptor-dependent mechanism.

Abstract: In the adult female rat, the densities of dendritic spines and synapses on hippocampal CA1 pyramidal cells are dependent upon the ovarian steroid estradiol; moreover, spine and synapse density fluctuate naturally as ovarian steroid levels vary across the estrous cycle. To determine whether the effects of estradiol on dendritic spine density require activation of specific neurotransmitter systems, we have treated animals concurrently with estradiol and one of four selective neurotransmitter receptor antagonists: MK 801, a noncompetitive NMDA receptor antagonist; CGP 43487, a competitive NMDA receptor antagonist; NBQX, an AMPA receptor antagonist; or scopolamine, a muscarinic receptor antagonist. Our results indicate that the effects of estradiol can be blocked by treatment with either of the NMDA receptor antagonists, but treatment with an AMPA or muscarinic receptor antagonist has no effect on spine density. Thus, we have concluded that estradiol exerts its effect on hippocampal dendritic spine density via a mechanism requiring activation specifically of NMDA receptors.

Axonal sprouting accompanies functional reorganization in adult cat striate cortex.

Abstract: Removal of sensory input from a focal region of adult neocortex can lead to a large reorganization of cortical topography within the deprived area during subsequent months. Although this form of functional recovery is now well documented across several sensory systems, the underlying cellular mechanisms remain elusive. Weeks after binocular retinal lesions silence a corresponding portion of striate cortex in the adult cat, this cortex again becomes responsive, this time to retinal loci immediately outside the scotoma. Earlier findings showed a lack of reorganization in the lateral geniculate nucleus and an inadequate spread of geniculocortical afferents to account for the cortical reorganization, suggesting the involvement of intrinsic cortical connections. We investigated the possibility that intracortical axonal sprouting mediates long-term reorganization of cortical functional architecture. The anterograde label biocytin was used to compare the density of lateral projections into reorganized and non-deprived cortex. We report here that structural changes in the form of axonal sprouting of long-range laterally projecting neurons accompany topographic remodelling of the visual cortex.

Human apolipoprotein A-I gene expression increases high density lipoprotein and suppresses atherosclerosis in the apolipoprotein E-deficient mouse

Abstract: Atherosclerosis is a complex disease with both genetic and environmental determinants. Apolipoprotein (Apo) E-deficient mice have been created that are highly susceptible to atherosclerosis. In order to assess the role of human apolipoprotein (hApo) A-I and high density lipoprotein (HDL) in atherosclerosis susceptibility, transgenic mice overexpressing the hApo A-I gene were crossed with Apo E-deficient mice. Apo E-/-, hApo A-I mice with two-fold elevation in HDL cholesterol have markedly diminished atherosclerosis with less fibroproliferative lesions by 8 months of age. A strong reciprocal relationship between HDL cholesterol levels and atherosclerosis was found with HDL levels accounting for 78% of the observed variance in mean lesion area. The effect of HDL on atherosclerosis resistance was independent of non-HDL cholesterol.

Superoxide Dismutase in Plants

Abstract: Superoxide dismutases (SODs) are metal-containing enzymes that catalyze the dismutation of superoxide radicals to oxygen and hydrogen peroxide. The enzyme has been found in all aerobic organisms examined where it plays a major role in the defense against toxic-reduced oxygen species, which are generated as byproducts of many biological oxidations. The generation of oxygen radicals can be further exacerbated during environmental adversity and consequently SOD has been proposed to be important for plant stress tolerance. In plants, three forms of the enzyme exist, as classified by their active site metal ion: copper/zinc, manganese, and iron forms. The distribution of these enzymes has been studied both at the subcellular level and at the phylogenic level. It is only in plants that all three different types of SOD coexist. Their occurrence in the different subcellular compartments of plant cells allows a study of their molecular evolution and the possibility of understanding why three functionally ...

A 13-amino-acid motif in the cytoplasmic domain of FcγRIIB modulates B-cell receptor signalling

Abstract: The Fc receptor on B lymphocytes, Fc gamma RIIB (beta 1 isoform), helps to modulate B-cell activation triggered by the surface immunoglobulin complex. Crosslinking of membrane immunoglobulin by antigen or anti-Ig F(ab')2 antibody induces a transient increase in cytosolic free Ca2+, a rise in inositol-3-phosphate, activation of protein kinase C, and enhanced protein tyrosine phosphorylation. Crosslinking Fc gamma RIIB with the surface immunoglobulin complex confers a dominant signal that prevents or aborts lymphocyte activation triggered through the ARH-1 motifs of the signal transduction subunits Ig-alpha and Ig-beta. Here we show that Fc gamma RIIB modulates membrane immunoglobulin-induced Ca2+ mobilization by inhibiting Ca2+ influx, without changing the pattern of tyrosine phosphorylation. A 13-amino-acid motif in the cytoplasmic domain of Fc gamma RIIB is both necessary and sufficient for this effect. Tyrosine at residue 309 in this motif is phosphorylated upon co-crosslinking with surface immunoglobulin; mutation of this residue aborts the inhibitory effect of Fc gamma RIIB. This inhibition is directly coupled to signalling mediated through Ig-alpha and Ig-beta as evidenced by chimaeric IgM/alpha and IgM/beta molecules. The 13-residue motif in Fc gamma RIIB controls lymphocyte activation by inhibiting a Ca2+ signalling pathway triggered through ARH-1 motifs as a result of recruitment of novel SH2-containing proteins that interact with this Fc gamma RIIB cytoplasmic motif.

Disruption of the HNF-4 gene, expressed in visceral endoderm, leads to cell death in embryonic ectoderm and impaired gastrulation of mouse embryos.

Abstract: Expression of HNF-4, a transcription factor in the steroid hormone receptor superfamily, is detected only in the visceral endoderm of mouse embryos during gastrulation and is expressed in certain embryonic tissues from 8.5 days of gestation. To examine the role of HNF-4 during embryonic development, we disrupted the gene in embryonic stem cells and found that the homozygous loss of functional HNF-4 protein was an embryonic lethal. Cell death was evident in the embryonic ectoderm at 6.5 days when these cells normally initiate gastrulation. As assessed by expression of Brachyury and HNF-3 beta, primitive streak formation and initial differentiation of mesoderm do occur, but with a delay of approximately 24 h. Development of embryonic structures is severely impaired. These results demonstrate that the expression of HNF-4 in the visceral endoderm is essential for embryonic ectoderm survival and normal gastrulation.

Apoptosis, but not necrosis, of infected monocytes is coupled with killing of intracellular bacillus Calmette-Guérin

Abstract: We have examined the effect of killing of host monocytes infected with bacillus Calmette-Guerin (BCG) on the viability of the intracellular mycobacteria. Peripheral blood monocytes were infected in vitro with a single bacillus per cell and maintained in culture for 6-8 d to allow the bacilli to replicate. Replicating viable BCG were found singly in perinuclear vacuoles bounded by tightly apposed lipid bilayers. Monocytes were then exposed to toxic mediators that induced killing of cells as evaluated by 51Cr release into the culture medium. Both hydrogen peroxide (H2O2) (an inducer of cell necrosis) and adenosine triphosphate (ATP4-) (an inducer of cell apoptosis) treatment killed infected monocytes. H2O2-induced killing had no effect on BCG viability. ATP-induced cell death was accompanied by DNA fragmentation and nuclear condensation. Apoptosis was associated with a swelling of the phagocytic vacuoles which became multibacillary and with a reduction of BCG viability as enumerated by colony-forming units.

Seasonal recruitment of hippocampal neurons in adult free-ranging black-capped chickadees

Abstract: Neurogenesis and neuronal replacement in a population of wild free-ranging animals are described Our subjects were adult black-capped chickadees (Parus atricapillus) Neuronal birth dates were determined by a single systemic injection of [3H]thymidine, followed by release of the bird and its recapture 6 or more weeks later Newly formed neurons appeared in the hippocampal complex during all times of year, but with a marked peak in the fall (October) New neurons were also added to the hippocampal complex of captive chickadees, but at levels only half as high as seen in the wild birds Neurons born at different times of the year lived for a few months and then disappeared We suggest that the neurons added are part of a process of neuronal replacement and that they are important for the acquisition of new spatial memories, a need that is particularly acute in the fall Hormonal and experiential factors may determine the rate of neuronal replacement

Functional immunoglobulin transgenes guide ordered B-cell differentiation in Rag-1-deficient mice.

Abstract: We have examined the regulatory role of the individual components of the immunoglobulin antigen receptor in B-cell development by transgenic complementation of Rag-1 deficient (Rag-1-) mice. Complementation with a membrane mu heavy chain (mu HC) gene allows progression of developmentally arrested Rag-1- pro-B-cells to the small pre-B cell stage, whereas the introduction of independently integrated mu HC and kappa light chain (kappa LC) transgenes promotes the appearance of peripheral lymphocytes which, however, remain unresponsive to external stimuli. Complete reconstitution of the B-cell lineage and the emergence of functionally nature Rag-1- peripheral B cells is achieved by the introduction of cointegrated heavy and light chain transgenes encoding an anti-H-2k antibody. This experimental system demonstrates the competence of the mu HC and kappa LC to direct and regulate the sequential stages of B-cell differentiation, defines the time at which negative selection of self-reactive B cells occurs, and shows that elimination of these cells occurs equally well in the absence of Rag-1 as in its presence. These data also support the hypothesis that Rag-1 directly participates in the V(D)J recombination process.

Lipopolysaccharide (LPS)-binding Protein Is Carried on Lipoproteins and Acts as a Cofactor in the Neutralization of LPS

Abstract: Lipoproteins isolated from normal human plasma can bind and neutralize bacterial lipopolysaccharide (LPS) and may represent an important mechanism in host defense against gram-negative septic shock. Recent studies have shown that experimentally elevating the levels of circulating high-density lipoproteins (HDL) provides protection against death in animal models of endotoxic shock. We sought to define the components of HDL that are required for neutralization of LPS. To accomplish this we have studied the functional neutralization of LPS by native and reconstituted HDL using a rapid assay that measures the CD14-dependent activation of leukocyte integrins on human neutrophils. We report here that reconstituted HDL particles (R-HDL), prepared from purified apolipoprotein A-I (apoA-I) combined with phospholipid and free cholesterol, are not sufficient to neutralize the biologic activity of LPS. However, addition of recombinant LPS binding protein (LBP), a protein known to transfer LPS to CD14 and enhance responses of cells to LPS, enabled prompt binding and neutralization of LPS by R-HDL. Thus, LBP appears capable of transferring LPS not only to CD14 but also to lipoprotein particles. In contrast with R-HDL, apoA-I containing lipoproteins (LpA-I) isolated from plasma by selected affinity immunosorption (SAIS) on an anti-apoA-I column, neutralized LPS without addition of exogenous LBP. Several lines of evidence demonstrated that LBP is a constituent of LpA-I in plasma. Passage of plasma over an anti-apoA-I column removed more than 99% of the LBP detectable by ELISA, whereas 31% of the LBP was recovered by elution of the column. Similarly, the ability of plasma to enable activation of neutrophils by LPS (LBP/Septin activity) was depleted and recovered by the same process. Furthermore, an immobilized anti-LBP monoclonal antibody coprecipitated apoA-I. The results described here suggest that in addition to its ability to transfer LPS to CD14, LBP may also transfer LPS to lipoproteins. Since LBP appears to be physically associated with lipoproteins in plasma, it is positioned to play an important role in the neutralization of LPS.

Identification of binding sites for transcription factors NF-kappa B and AP-2 in the promoter region of the human heme oxygenase 1 gene

Abstract: Heme oxygenase (HO) is the rate-limiting enzyme in heme catabolism and its activity is induced by many agents, including its substrate heme, heavy metals, UV radiation, and other injurious oxidant conditions. We examined the presence of several regulatory elements in the promoter region of the human HO-1 gene which could possibly account for its induction in response to diverse agents or influences. Heme treatment increased both HO activity and HO-1 mRNA in the human erythroleukemic cell line K562. Electrophoretic mobility-shift assays of nuclear protein extracts from heme-treated and control cells with specific oligonucleotide probes containing binding sites for known transcription factors, including AP-1, AP-2, Sp1, NF-kappa B, CTF/NF1, TFIID, OKT1, and CREB, and oligonucleotides containing serum-, metal-, and glucocorticoid-responsive elements demonstrated a specific and marked increase in the NF-kappa B and AP-2 transcription factors and, to a lesser extent, an increase in AP-1. No significant increase in other transcription factors over the control, untreated cells was observed. DNase I footprint assays using purified transcription factors revealed the presence of NF-kappa B and AP-2 binding sites in the proximal part of the promoter region of the human HO-1 gene. Moreover, nucleotide sequence analysis of the HO-1 promoter region showed that the protected regions encompassed NF-kappa B and AP-2 consensus binding sites. The presence of regulatory sequences for the binding of transcription factors such as NF-kappa B and AP-2, whose activation is associated with the immediate response of the cell to an injury, may be an indication of the important role which HO-1 may play in defense mechanisms against tissue injury.

Multiple-antibiotic-resistant Pathogenic Bacteria -- A Report on the Rockefeller University Workshop

Abstract: Last year, a small group of scientists, physicians, and public health experts gathered at Rockefeller University for a one-day workshop to discuss the accelerating spread of bacterial pathogens resistant to antimicrobial agents and whether multiresistant bacteria pose a threat to public health in the United States. This report is based on the conclusions of that workshop. After a half-century of virtually complete control over microbial disease in the developed countries, the 1990s have brought a worldwide resurgence of bacterial and viral diseases1. An important factor in this phenomenon is the acquisition of antibiotic-resistance genes by virtually all major bacterial . . .