GGA1 acts as a spatial switch altering amyloid precursor protein trafficking and processing

The beta-amyloid (Abeta) precursor protein (APP) is cleaved sequentially by beta-site of APP-cleaving enzyme (BACE) and gamma-secretase to release the Abeta peptides that accumulate in plaques in Alzheimer's disease (AD). GGA1, a member of the Golgi-localized gamma-ear-containing ARF-binding (GGA) protein family, interacts with BACE and influences its subcellular distribution. We now report that overexpression of GGA1 in cells increased the APP C-terminal fragment resulting from beta-cleavage but surprisingly reduced Abeta. GGA1 confined APP to the Golgi, in which fluorescence resonance energy transfer analyses suggest that the proteins come into close proximity. GGA1 blunted only APP but not notch intracellular domain release. These results suggest that GGA1 prevented APP beta-cleavage products from becoming substrates for gamma-secretase. Direct binding of GGA1 to BACE was not required for these effects, but the integrity of the GAT (GGA1 and TOM) domain of GGA1 was. GGA1 may act as a specific spatial switch influencing APP trafficking and processing, so that APP-GGA1 interactions may have pathophysiological relevance in AD.     

Modulation of the crayfish swimmeret rhythm by octopamine and the neuropeptide proctolin

1. The swimmeret system can be excited by perfusing the neuropeptide proctolin through the isolated ventral nerve cord of the crayfish. Previously silent preparations begin to generate a characteristic motor pattern, the swimmeret rhythm, in the nerves that innervate the swimmerets. The response to proctolin is dose dependent and reversible. The threshold concentration of proctolin perfused through the ventral artery is approximately 10(-8) M. The EC50 is 1.6 X 10(-6) M. 2. Proctolin-induced motor patterns have periods and phases similar to those of spontaneously generated motor patterns. The durations of the bursts of impulses in power-stroke motor neurons generated in the presence of proctolin are, however, significantly longer than those that occur during spontaneous activity. 3. DL-Octopamine inhibits the swimmeret system, both when the system is spontaneously active and when it has been excited by proctolin. The inhibition by octopamine is dose dependent and reversible. The threshold for inhibition is approximately 10(-6) M, and the EC50 is approximately 5 X 10(-5) M. 4. Octopamine's effect is mimicked by its agonists, synephrine and norepinephrine. Synephrine has a lower threshold concentration than does octopamine, but norepinephrine is much less effective than octopamine. 5. Octopamine's inhibition is partially blocked by an antagonist, phentolamine. 6. Phentolamine also blocks inhibition of the swimmeret system by inhibitory command interneurons. This block is dose dependent and can be partially overcome by stimulating the command interneurons at higher frequencies. 7. Perfusion with 11 other suspected crustacean neurotransmitters and transmitter analogues did not similarly excite or inhibit the swimmeret system, so we suggest that proctolin and octopamine are transmitters used by the neurons that normally control expression of the swimmeret rhythm.     

大鼠胸腺降黑素受体的鉴定及生物学特性

应用放射配体结合法证实大鼠胸腺内存在降黑素特异结合部位，该结合位点可以满足特异结合部位的基本条件：１．低结合容量；２．高亲和力；３．可饱和性；４．可逆性；５．对降黑素高度特异性。此外，该特异结合位点具昼夜节律；亚细胞分布的研究表明以细胞核含量最高，线粒体次之，并具有年龄依赖性降低，以出生时最高。     

Expression of human choriogonadotropin-like material in coagulase-negative Staphylococcus species.

We identified 101 coagulase-negative Staphylococcus strains obtained from different laboratories, the American Type Culture Collection, and our collection, isolated from 23 patients with overt cancer and 34 normal individuals through Kloos and Schleifer conventional methods and the Staph-Ident staphylococcal system (Analytab Products, Plainview, N.Y.). In 40 strains, identity was further verified by DNA-DNA hybridization techniques. Identification revealed 39 S. epidermidis, 22 S. hominis, 8 S. haemolyticus, 9 S. capitis, 5 S. warneri, 5 S. cohnii, 8 S. saprophyticus, and 5 S. xylosus strains, all resident species found in humans. All bacteria were tested for the expression of human choriogonadotropin (hCG)-like material by the indirect fluorescein and peroxidase immunocytochemical labeling techniques by using specific antisera to the whole hormone, to its alpha and beta subunits, to the hCG beta COOH-terminal peptide, and to a monoclonal antibody to the hCG beta. The results demonstrated that the isolates from cancer patients were not unique bacteria, as has been postulated by others; the expression of immunoreactive hCG-like material is a strain, not a species, characteristic; not every bacterial strain isolated from a cancer patient is able to express the material; hCG-producing bacteria do not necessarily indicate the presence of active disease; 20% of the strains that we studied revealed a clonal variation of the expression of hCG-like material or its subunits or both as well as a variable expression of a single hCG epitope, an observation similar to that described for malignant cells; and a specific antiserum to the whole hormone with a high affinity and high sensitivity for immunocytochemistry can be a reliable reagent for screening purposes.     

ROCK inhibitor (Y27632) increases apoptosis and disrupts the actin cortical mat in embryonic avian corneal epithelium.

The embryonic chicken corneal epithelium is a unique tissue that has been used as an in vitro epithelial sheet organ culture model for over 30 years (Hay and Revel [1969] Fine structure of the developing Avian cornea. Basel, Switzerland: S. Karger A.G.). This tissue was used to establish that epithelial cells could produce extracellular matrix (ECM) proteins such as collagen and proteoglycans (Dodson and Hay [1971] Exp Cell Res 65:215-220; Meier and Hay [1973] Dev Biol 35:318-331; Linsenmayer et al. [1977] Proc Natl Acad Sci U S A 74:39-43; Hendrix et al. [1982] Invest Ophthalmol Vis Sci 22:359-375). This historic model was also used to establish that ECM proteins could stimulate actin reorganization and increase collagen synthesis (Sugrue and Hay [1981] J Cell Biol 91:45-54; Sugrue and Hay [1982] Dev Biol 92:97-106; Sugrue and Hay [1986] J Cell Biol 102:1907-1916). Our laboratory has used the model to establish the signal transduction pathways involved in ECM-stimulated actin reorganization (Svoboda et al. [1999] Anat Rec 254:348-359; Chu et al. [2000] Invest Ophthalmol Vis Sci 41:3374-3382; Reenstra et al. [2002] Invest Ophthalmol Vis Sci 43:3181-3189). The goal of the current study was to investigate the role of ECM in epithelial cell survival and the role of Rho-associated kinase (p160 ROCK, ROCK-1, ROCK-2, referred to as ROCK), in ECM and lysophosphatidic acid (LPA) -mediated actin reorganization. Whole sheets of avian embryonic corneal epithelium were cultured in the presence of the ROCK inhibitor, Y27632 at 0, 0.03, 0.3, 3, or 10 microM before stimulating the cells with either collagen (COL) or LPA. Apoptosis was assessed by Caspase-3 activity assays and visualized with annexin V binding. The ROCK inhibitor blocked actin cortical mat reformation and disrupted the basal cell lateral membranes in a dose-dependent manner and increased the apoptosis marker annexin V. In addition, an in vitro caspase-3 activity assay was used to determine that caspase-3 activity was higher in epithelia treated with 10 microM Y-27632 than in those isolated without the basal lamina or epithelia stimulated with fibronectin, COL, or LPA. In conclusion, ECM molecules decreased apoptosis markers and inhibiting the ROCK pathway blocked ECM stimulated actin cortical mat reformation and increased apoptosis in embryonic corneal epithelial cells.     

Exposure to Organic Dust Causes Activation of Human Plasma Complement Factors C3 and B and the Synthesis of Factor C3 by Lung Epithelial Cells <Emphasis Type="Italic">In Vitro</Emphasis>

Exposure in swine confinement buildings induces an intense airway inflammation. Twenty-two volunteers, of whom eleven wore a half-mask, were exposed for 3 hr in a swine barn. Blood samples were drawn before and after exposure. The ratio C3b/totalC3 in plasma decreased from 6.8 to 5.0% (p = 0.02) without mask and from 6.6 to 5.9% (p = 0.01) with mask (p = 0.67 between groups). The ratio Bb/totalB decreased from 14.5 to 13.5% (p < 0.01) without and 14.6–13.3% (p = 0.09) with mask (p = 0.25 between groups). Epithelial cells (A549) incubated up to 24 hr with 0.1 mg/mL dust suspensions were analysed for C3, IL-6 and IL-8 secretion. Cumulative C3 synthesis of dust stimulated cell cultures was 43,000 pg/mL compared to 25,000 pg/mL in unstimulated cells. Cumulative dust-induced IL-6 and IL-8 secretion was 200 and 3000 pg/mL, respectively and below detection in unstimulated cells.The activation of complement in vivo and induced C3 synthesis by epithelial cells suggests a role of complement in the airway reaction to organic dust exposure.     

Hypoxia down-regulates placenta growth factor, whereas fetal growth restriction up-regulates placenta growth factor expression: molecular evidence for "placental hyperoxia" in intrauterine growth restriction

Early placental development occurs in an environment of relative hypoxia. Hypoxia promotes angiogenesis and up-regulates vascular endothelial growth factor (VEGF) expression while it down-regulates placenta growth factor (PIGF) that possess 53% homology with VEGF. Morphological studies show poor placental vascular development and an increase in the mitotic index of cytotrophoblasts in intrauterine growth restriction (IUGR). We hypothesized that the reported relatively high oxygen level in the intervillous space in contact with IUGR placental villi will limit angiogenesis by changes in VEGF and PIGF expression and function. Western immunoblot analysis demonstrates a diametric expression of PIGF and VEGF proteins throughout pregnancy with PIGF levels increasing and VEGF levels decreasing, consistent with placental oxygenation. In IUGR placentae, the ratio of PIGF/GAPDH mRNA was increased by 2.3-fold (p < 0.03) and PIGF protein levels were also increased, (p < 0.05) as compared with gestationally-matched normal placentae. PIGF mRNA and protein were localized to the trophoblast bilayer and villous mesenchyme of the human placenta throughout gestation. In vitro studies demonstrated that increasing oxygen tension (hyperoxia) up-regulated PIGF protein in term placental villous explants, whereas hypoxic culture of a term trophoblast choriocarcinoma cell line (BeWo) down-regulated PIGF mRNA and protein and VEGFR-1 (Flt-1) autophosphorylation. The addition of PIGF-1 to a spontaneously transformed first trimester cytotrophoblast cell line stimulated DNA synthesis while PIGF-2 had little effect. VEGF and PIGF exert their biological actions by means of a common receptor VEGFR-1. In the first trimester trophoblast cells, PIGF-1 increased the association of phosphorylated extracellular signal-related kinase (ERK) with VEGFR-1 immunoprecipitates while both PIGF-1 and PIGF-2 also potentiated endogenous VEGF mediated association of phosphorylated extracellular related kinase (ERK) with VEGFR-2 (KDR). More importantly, the addition of PIGF-1 had little effect while PIGF-2 inhibited cell growth in cultured endothelial cells derived from human umbilical vein. Nitric oxide (NO) is reported to promote angiogenesis and PIGF-2 inhibited the basal release of NO from the first trimester trophoblast. The tissue expression and functional studies support the hypothesis of "placental hyperoxia" in early-onset IUGR because hypoxia down-regulates trophoblast PIGF levels, PIGF expression is increased in IUGR, and PIGF-2 inhibits endothelial cell growth. Taken together, these changes provide a cellular explanation for the observed poor angiogenesis in the pathogenesis of IUGR and show that the two PIGF isoforms may modulate trophoblast and endothelial cell function differently, possibly through potentiation of VEGF mediated activation of VEGF-2.     

Immunohistochemical localization of a choriogonadotropin-like protein in bacteria isolated from cancer patients.

By the use of specific antibody to human chorionic gonadotropin (CG) as well as to its beta-subunit, and the application of the indirect fluorescein-labeled and peroxidase-labeled antibody techniques, we have demonstrated the presence of a membrane (wall)-associated CG-similar immunoreactive protein in 15 strains of bacteria isolated from tissues of patients bearing malignant neoplasms. These microorganisms were classified as S. epidermidis, (12) E. coli (2), and a single strain of P. maltophilia (ATCC 13637). The absence of the CG-like antigen in other "cancer associated bacteria", Streptococcus faecalis (ATCC 12818) and Pseudomonas aeruginosa (from patient with cancer of colon), demonstrated that not every "cancer associated bacteria" has the capability to synthesize the trophoblastic-like protein. The negative results obtained with a number of "noncancer control" bacteria of known origin, obtained from ATCC and from clinical samples, strongly supported the idea that the existance of these CG-like protein producing microorganisms is not a ubiquitous finding. The demonstration of a de novo bacterial biosynthesis of a protein having similar antigenic and biophysical properties to those of the human trophoblastic hormone, has great biological implications, especially if its biosynthesis is proven only in bacterial strains growing in the presence of cancer cells in which we have already demonstrated the presence of a similar antigen. The explanation of the phenomenon is unknown. Because of their origin, the potential of "genetic exchange" with subsequent expression of the mammalian gene by the bacterial cells becomes a possibility. It is also possible that the gene coding for the CG-like protein is normally present but inactive or repressed in all bacteria.