RE-EXAMINATION OF FERTILIZATION INHIBITION STUDIES WITH ANTI-FROG-EGG-JELLY ANTIBODIES: EXPERIMENTS WITH NON-PRECIPITATING ANTIBODIES

Antisera were prepared in rabbits against oviducal and egg-jellies of the frog, Rana japonica . Gamma-globulin fractions from the antisera were degraded to a univalent, non-precipitating form by a papain digestion-reduction procedure. Agar diffusion analyses proved that the digested antibody fragments were inhibitory to the precipitin reaction of undigested, multivalent antibodies with jelly antigens. The treatment of unfertilized eggs with undigested, multivalent antibodies resulted in a significant loss of egg-fertilizability. In contrast, treatment with the univalent fragments of antibodies did not affect egg-fertilizability, similarly to the treatment with both univalent and multivalent γ-globulins from control, non-immune sera. Fertilization was inhibited in large measure when unfertilized eggs were subjected to a dual treatment with univalent antibodies and γ-globulins from anti-rabbit γ-globulin sheep serum. The inhibition of fertilization in the above experiments was always accompanied by the formation of a precipitation layer at the surface of the jelly envelopes. It is concluded that the failure of fertilization in the multivalent antibody-treated eggs results from a secondary effect rather than a specific blocking of a sperm-jelly interaction essential for fertilization.     

VIROSOMES IN VACCINE DEVELOPMENT: INDUCTION OF CYTOTOXIC T LYMPHOCYTE ACTIVITY WITH VIROSOME-ENCAPSULATED PROTEIN ANTIGENS

ABSTRACT

Virosomes are reconstituted viral envelopes which lack the genetic material but retain the cell entry and membrane fusion characteristics of the virus they are derived from. Thus, influenza virosomes are taken up by cells via receptor-mediated endocytosis, which directs the particles to the endosomal cell compartment. Subsequently, the virosomal membrane fuses with the endosomal membrane induced by the mildly acidic pH within the endosomes. This fusion process establishes continuity between the lumen of the virosome and the cell cytosol. Upon interaction of virosomes with antigen-presenting cells (APCs), protein antigens encapsulated within virosomes will be delivered to the cell cytosol, and thus, into the MHC class I presentation pathway. Indeed, virosome-mediated delivery of antigens in vivo results in efficient priming of a class I MHC-restricted cytotoxic T lymphocyte (CTL) response.     

蚕豆田潜蝇姬小蜂白昼活动规律

本文就蚕豆上潜蝇姬小蜂白昼活动规律进行了研究,在午间13:00-14:00时气温最高时,为潜蝇姬小蜂的活动高峰期,种群(Y)和温度(X)的白昼活动模式为Y=-2.41+1.38x-0.09x2,r=0.7221··;在蚕豆成熟时,潜蝇姬小蜂种群数量最大.     

CORRELATION OF THE FUNCTIONAL ACTIVITY OF THE BRAIN WITH METABOLIC PARAMETERS—III*

1. The residual protein content per unit DNA of the tissue rises markedly in occipital cortex, cerebral cortex, tectum and hippocampus of the developing rabbits in days following the opening of the eyes. The change is much less marked in cerebellar cortex during this period. 2. Prior blinding of the animals diminishes significantly the rise in proteins of the occipital cortex. The effect of blinding on the expected rise in proteins of other areas is not significant. 3. Exposure of adult animals to light stimulates the incorporation of dl -[1-¹⁴C]- valine and [U-¹⁴C]glucose into proteins of the occipital cortex as compared to animals kept in the dark room.     

Two Sides of MGP Null Arterial Disease: CHONDROGENIC LESIONS DEPENDENT ON TRANSGLUTAMINASE 2 AND ELASTIN FRAGMENTATION ASSOCIATED WITH INDUCTION OF ADIPSIN*

Mutations in matrix Gla protein (MGP) have been correlated with vascular calcification. In the mouse model, MGP null vascular disease presents as calcifying cartilaginous lesions and mineral deposition along elastin lamellae (elastocalcinosis). Here we examined the mechanisms underlying both of these manifestations. Genetic ablation of enzyme transglutaminase 2 (TG2) in Mgp^−/− mice dramatically reduced the size of cartilaginous lesions in the aortic media, attenuated calcium accrual more than 2-fold, and doubled longevity as compared with control Mgp^−/− animals. Nonetheless, the Mgp^−/−;Tgm2^−/− mice still died prematurely as compared with wild-type and retained the elastocalcinosis phenotype. This pathology in Mgp^−/− animals was developmentally preceded by extensive fragmentation of elastic lamellae and associated with elevated serine elastase activity in aortic tissue and vascular smooth muscle cells. Systematic gene expression analysis followed by an immunoprecipitation study identified adipsin as the major elastase that is induced in the Mgp^−/− vascular smooth muscle even in the TG2 null background. These results reveal a central role for TG2 in chondrogenic transformation of vascular smooth muscle and implicate adipsin in elastin fragmentation and ensuing elastocalcinosis. The importance of elastin calcification in MGP null vascular disease is highlighted by significant residual vascular calcification and mortality in Mgp^−/−;Tgm2^−/− mice with reduced cartilaginous lesions. Our studies identify two potential therapeutic targets in vascular calcification associated with MGP dysfunction and emphasize the need for a comprehensive approach to this multifaceted disorder.     

Eyespot Disease of Sugarcane : INDUCTION OF HOST-SPECIFIC TOXIN AND ITS INTERACTION WITH LEAF CELLS

Helminthosporium sacchari produces a toxin which is responsible for the symptoms of eyespot disease in Saccharum officinarum. A rapid and highly repeatable bioassay based on increase in conductivity of tissue leachates showed that the interaction of toxin with sugarcane obeys Michaelis-Menten hyperbolic saturation kinetics. There was no evidence for positive or negative cooperation interaction. Resistant and susceptible cultivars of sugar cane had distinctive conductivity characteristics. Co-cultures of H. sacchari and suspension cultures of sugarcane gave up to a 4,000-fold increase in toxin production.     

SYNAPTOSOMAL TYROSINE HYDROXYLATION IN THE RAT BRAIN: COMPARISON OF ACTIVITY FROM HIPPOCAMPUS AND HYPOTHALAMUS WITH ACTIVITY FROM STRIATUM

Abstract— A modified tritium release assay for the measurement of synaptosomal tyrosine hydroxyl-ation. with a sensitivity suitable for use on areas of the rat brain with a low density of catecholamine terminals. is described. The apparent K_m , for tyrosine hydroxylase in the hippocampus was 9.3 μM. in the hypothalamus 6.1 μM and in the striatum 9.9 μM Preparations from all three regions showed a pH optimum of 6.0–6.2, and the activities were reduced to a small % of control by synaptosomal disruption. 3-iodotyrosine. noradrenaline and reserpine. Membrane depolarization at a pH of 6.1 did not elevate tyrosine hydroxylation rates in any of the regions studied, although striatal tyrosine hy-droxylation rates were elevated at a pH of 7.2 by 55 mM-K⁺. The addition of dibutyryl cyclic AMP (0.5 mM) to the medium produced a 20-30% elevation of the rates of hydroxylation in all three regions studied: addition of tetrahydrobiopterin (0.2 mM) elevated hydroxylation rates in the hypothalamus and striatum. These results indicate that many characteristics of tyrosine hydroxylase from the three regions are similar. In each case the enzyme is apparently sensitive to end-product inhibition and to cyclic AMP activation.     

Streptococcus sanguinis Class Ib Ribonucleotide Reductase: HIGH ACTIVITY WITH BOTH IRON AND MANGANESE COFACTORS AND STRUCTURAL INSIGHTS*

Streptococcus sanguinis is a causative agent of infective endocarditis. Deletion of SsaB, a manganese transporter, drastically reduces S. sanguinis virulence. Many pathogenic organisms require class Ib ribonucleotide reductase (RNR) to catalyze the conversion of nucleotides to deoxynucleotides under aerobic conditions, and recent studies demonstrate that this enzyme uses a dimanganese-tyrosyl radical (Mn^III₂-Y^•) cofactor in vivo. The proteins required for S. sanguinis ribonucleotide reduction (NrdE and NrdF, α and β subunits of RNR; NrdH and TrxR, a glutaredoxin-like thioredoxin and a thioredoxin reductase; and NrdI, a flavodoxin essential for assembly of the RNR metallo-cofactor) have been identified and characterized. Apo-NrdF with Fe^II and O₂ can self-assemble a diferric-tyrosyl radical (Fe^III₂-Y^•) cofactor (1.2 Y^•/β₂) and with the help of NrdI can assemble a Mn^III₂-Y^• cofactor (0.9 Y^•/β₂). The activity of RNR with its endogenous reductants, NrdH and TrxR, is 5,000 and 1,500 units/mg for the Mn- and Fe-NrdFs (Fe-loaded NrdF), respectively. X-ray structures of S. sanguinis NrdI_ox and Mn^II₂-NrdF are reported and provide a possible rationale for the weak affinity (2.9 μm) between them. These streptococcal proteins form a structurally distinct subclass relative to other Ib proteins with unique features likely important in cluster assembly, including a long and negatively charged loop near the NrdI flavin and a bulky residue (Thr) at a constriction in the oxidant channel to the NrdI interface. These studies set the stage for identifying the active form of S. sanguinis class Ib RNR in an animal model for infective endocarditis and establishing whether the manganese requirement for pathogenesis is associated with RNR.     

THE INDUCTION OF MELANIZATION IN GOLDFISH SCALES WITH ACTH, IN VITRO : Cellular and Subcellular Changes

The induction of melanization in xanthic goldfish scales with ACTH in vitro has been studied by light and electron microscopy utilizing ammoniated silver nitrate staining of premelanin and melanin. The melanized cells (melanophores and melanocytes) and the yellow pigmented cells (lipophores and the newly described lipocytes) were found to possess many similarities at the levels of cellular and subcellular structure. The latter cells contain characteristic cytoplasmic bodies which react positively to the premelanin stain. Changes accompanying ACTH stimulation of goldfish scales in tissue culture suggest that these bodies in the lipocytes and lipophores can become melanized. Electron micrographs illustrate the intermediate staining of newly formed melanin granules in an induced melanocyte and the appearance of a transitional melanolipophore. It is postulated that ACTH can promote the association of the enzyme tyrosinase with the preformed structure of unmelanized granules.     

PHYTOPLANKTON PLASMA MEMBRANE REDOX ACTIVITY: EFFECT OF IRON LIMITATION AND INTERACTION WITH PHOTOSYNTHESIS1

Phytoplankton plasma membrane electron transport activity was determined by monitoring the reduction of the impermeant artificial electron acceptor ferricyanide in a range of diatoms. The results revealed that constitutive plasma membrane electron transport activity of marine diatoms is high compared with chlorophytes and higher plant cells. Diatom plasma membrane electron transport activity was not significantly increased by iron limitation. This lack of induction on iron limitation indicates that diatoms have an iron acquisition strategy that is distinct from chlorophytes and the dicotyledon higher plants that exhibit marked increases in plasma membrane ferricyanide reductase activity on iron limitation. The interaction of the constitutive plasma membrane electron transport with photosynthesis was also investigated. We found that 1) ferricyanide reduction at the plasma membrane was progressively inhibited in response to increasing irradiances; 2) the presence of extracellular ferricyanide, but not the reduced couple ferrocyanide, caused a marked inhibition of carbon fixation at high irradiance; and 3) extracellular electron acceptors ferricyanide and hexachloroiridate (but not ferrocyanide) induced an immediate and reversible decrease in fluorescence yields (F_o and F_m). The extent to which extracellular electron acceptors affected CO₂ fixation, F_o, and F_m was related to the level of constitutive ferricyanide reductase activity, the species with highest ferricyanide reduction rates being most sensitive. The data suggest that consumption of electrons and/or reductant at the plasma membrane by external acceptors may compete directly with CO₂ fixation for electrons, alter cytosolic‐chloroplast redox poise, and/or induce a redox‐signaling cascade that alters photosynthetic metabolism.     

INDUCTION OF CHROMOSOME MOTION IN THE GLYCEROL-ISOLATED MITOTIC APPARATUS: NUCLEOTIDE SPECIFICITY AND EFFECTS OF ANTIDYNEIN AND MYOSIN SERA ON THE MOTION*

Chromosome motion in glycerol-isolated mitotic apparatus (MA) of sea urchin and starfish eggs was investigated with respect to nucleotide specificity and the effects of antisera against tryptic fragment (Fragment A) of flagellar dynein and starfish egg myosin. The motion was highly specific for ATP. GTP, ITP, CTP, UTP, and ADP caused no displacement of the chromosomes towards the poles. The anti-Fragment A serum completely inhibited chromosome motion in the MA of the sea urchin egg, while antiserum against starfish egg myosin as well as its γ-globulin fraction did not inhibit the motion in the isolated MA of the starfish egg, suggesting that chromosome motion depends upon dynein-microtubule but not upon myosin-actin interaction. In addition, colchicine completely suppressed the chromosome motion in vitro.     

ScFv Antibody-induced Translocation of Cell-surface Heparan Sulfate Proteoglycan to Endocytic Vesicles: EVIDENCE FOR HEPARAN SULFATE EPITOPE SPECIFICITY AND ROLE OF BOTH SYNDECAN AND GLYPICAN*

Cellular uptake of several viruses and polybasic macromolecules requires the expression of cell-surface heparan sulfate proteoglycan (HSPG) through as yet ill defined mechanisms. We unexpectedly found that among several cell-surface-binding single chain variable fragment (scFv) anti-HS antibody (αHS) clones, only one, AO4B08, efficiently translocated macromolecular cargo to intracellular vesicles through induction of HSPG endocytosis. Interestingly, AO4B08-induced PG internalization was strictly dependent on HS 2-O-sulfation and appeared independent of intact N-sulfation. AO4B08 and human immunodeficiency virus (HIV)-Tat, i.e. a well known cell-penetrating peptide, were shown to compete for the internalizing PG population. To obtain a more detailed characterization of this pathway, we have developed a procedure for the isolation of endocytic vesicles by conjugating AO4B08 with superparamagnetic nanoparticles. [³⁵S]sulfate-labeled HSPG was found to accumulate in isolated, AO4B08-containing vesicles, providing the first biochemical evidence for intact HSPG co-internalization with its ligand. Further analysis revealed the existence of both syndecan, i.e. a transmembrane HSPG, and glycosyl-phosphatidyl-inositol-anchored glypican in purified vesicles. Importantly, internalized syndecan and glypican were found to co-localize in AO4B08-containing vesicles. Our data establish HSPGs as true internalizing receptors of macromolecular cargo and indicate that the sorting of cell-surface HSPG to endocytic vesicles is determined by a specific HS epitope that can be carried by both syndecan and glypican core protein.     

Crown Gall Tumor Disc Bioassay: A POSSIBLE AID IN THE DETECTION OF COMPOUNDS WITH ANTITUMOR ACTIVITY

Seventeen samples consisting of purified compounds and various ethanol extracts from plant sources were tested for activity on the initiation of crown gall tumors on potato discs. The results demonstrated definite correlation between the ability of these samples to inhibit the formation of crown gall tumors and their activity on the P388 leukemia system in mice. Samples showing only cytotoxic effects in KB cell cultures did not affect tumor initiation in our system. The active materials had no effects on bacterial viability or on the ability of the bacteria to attach to a tumorbinding site.     

Isoform-selective Inhibition of Facilitative Glucose Transporters: ELUCIDATION OF THE MOLECULAR MECHANISM OF HIV PROTEASE INHIBITOR BINDING*

Pharmacologic HIV protease inhibitors (PIs) and structurally related oligopeptides are known to reversibly bind and inactivate the insulin-responsive facilitative glucose transporter 4 (GLUT4). Several PIs exhibit isoform selectivity with little effect on GLUT1. The ability to target individual GLUT isoforms in an acute and reversible manner provides novel means both to investigate the contribution of individual GLUTs to health and disease and to develop targeted treatment of glucose-dependent diseases. To determine the molecular basis of transport inhibition, a series of chimeric proteins containing transmembrane and cytosolic domains from GLUT1 and GLUT4 and/or point mutations were generated and expressed in HEK293 cells. Structural integrity was confirmed via measurement of N-[2-[2-[2-[(N-biotinylcaproylamino)ethoxy)ethoxyl]-4-[2-(trifluoromethyl)-3H-diazirin-3-yl]benzoyl]-1,3-bis(mannopyranosyl-4-yloxy)-2-propylamine (ATB-BMPA) labeling of the chimeric proteins in low density microsome fractions isolated from stably transfected 293 cells. Functional integrity was assessed via measurement of zero-trans 2-deoxyglucose (2-DOG) uptake. ATB-BMPA labeling studies and 2-DOG uptake revealed that transmembrane helices 1 and 5 contain amino acid residues that influence inhibitor access to the transporter binding domain. Substitution of Thr-30 and His-160 in GLUT1 to the corresponding positions in GLUT4 is sufficient to completely transform GLUT1 into GLUT4 with respect to indinavir inhibition of 2-DOG uptake and ATB-BMPA binding. These data provide a structural basis for the selectivity of PIs toward GLUT4 over GLUT1 that can be used in ongoing novel drug design.