Effects of L-Glutamate Transport Inhibition by a Conformationally Restricted Glutamate Analogue (2S,1'S,2'R)-2-(Carboxycyclopropyl)Glycine (L-CCG III) on Metabolism in Brain Tissue In Vitro Analysed by NMR Spectroscopy

(2S,1'S,2'R)-2-(Carboxycyclopropyl)glycine (L-CCG III) was a substrate of Na⁺-dependent glutamate transporters (GluT) in Xenopus laevis oocytes (IC₅₀ 13 and 2 M for, respectively, EAAT 1 and EAAT 2) and caused an apparent inhibition of [³H]L-glutamate uptake in mini-slices of guinea pig cerebral cortex (IC₅₀ 12 M). In slices (350 M) of guinea pig cerebral cortex, 5 M L-CCG III increased both the flux of label through pyruvate carboxylase and the fractional enrichment of glutamate, GABA, glutamine and lactate, but had no effect on total metabolite pool sizes. At 50 M L-CCG III decreased incorporation of ¹³C from [3-¹³C]-pyruvate into glutamate C4, glutamine C4, lactate C3 and alanine C3. The total metabolite pool sizes were also decreased with no change in the fractional enrichment. Furthermore, L-CCG III was accumulated in the tissue, probably via GluT. At lower concentration, L-CCG III would compete with L-glutamate for GluT and the changes probably reflect a compensation for the missing L-glutamate. At 50 M, intracellular L-CCG III could reach > 10 mM and metabolism might be affected directly.     

Gliding defective mutant cell lines ofChlamydomonas moewusii exhibit alterations in a 240 kDa surface-exposed flagellar glycoprotein

Summary TheChlamydomonas flagellar surface exhibits a number of dynamic properties associated with whole cell locomotion and the mating process. In this report, we quantitate the ability of a series of gliding defective mutant cell lines (Lewin 1982) to move polystyrene microspheres along their flagellar surface and describe alterations in the flagellar surfaces of three of these cell lines (fg-2, fg-3 and fg-7). Although all three of these mutant cell strains exhibit less than 16% of the control level of microsphere movement, they differ from each other and the parental cell line (M 475) in the level of flagellar surface adhesiveness as judged by the binding of polystyrene microspheres. SDS-polyacrylamide gel analysis of purified whole flagella from the nongliding mutant cell strains and M 475 demonstrates a correlation between the amount of a surface exposed glycoprotein and the level of flagellar surface adhesiveness. This surface exposed glycoprotein binds the lectin concanavalin A and has an apparent molecular weight of 240 kDa. Strains with low levels of flagellar surface adhesiveness (fg-3 and fg-7) exhibit a low amount of this glycoprotein while the strain with a high level of adhesiveness (fg-2) has an elevated amount of this glycoprotein relative to the parental strain, suggesting that this 240 kDa glycoprotein may be responsible for the adhesive properties of the flagellar surface. Concanavalin A inhibits microsphere binding to the flagellar surface, suggesting that the carbohydrate component of the 240 kDa glycoprotein may be involved in flagellar surface adhesiveness. Biotinylation of surface-exposed flagellar proteins demonstrates differences in the surfaces of these mutant cell lines, especially in terms of the amount of surface labelling of the 240 kDa flagellar glycoprotein. A rabbit polyclonal antibody (designated P-19) that binds to the flagellar surface and recognizes the 240 kDa glycoprotein on Western blots confirms the altered level of this glycoprotein in the mutant cell lines. The results of these experiments suggest that the major flagellar glycoprotein ofC. moewusii may be involved in adhesion of polystyrene microspheres to the flagellar surface and presumably also in the adhesive interaction of the flagellar surface with a solid substrate, which is a necessary prerequisite for the expression of gliding motility.Abbreviations BSA bovine serum albumin - DAB 3,3-diaminobenzidine - HRP horseradish peroxidase - kDa kilodaltons - LBB lectin blot buffer - NHS-LC biotin sulfosuccinimidyl 6-(biotinamido) hexanoate - PBS phosphate buffered saline - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

Physicochemical and in situ observations on the adhesion of gliding bacteria to surfaces

The ability of Flexibacter BH3 to adhere to solid surfaces and to overcome the horizontal drag involved in gliding across the surfaces was considered in terms of the Stefan adhesion principle. The extracellular slime produced by Flexibacter BH3 was suitable as a Stefan adhesive because it exhibited viscous properties characteristic of a linear colloid, increasing the adhesiveness of the bacterium but allowing translational motion across the surface. The water-soluble slime was a glycoprotein, containing glucose, fucose, galactose and some uronic acid. Vesicles and tubules on the outer surface of Flexibacter BH3 possessed trilaminar membranes, contained 2-keto-3-deoxyoctonate (KDO), and showed identity with phenol-extracted lipopolysaccharide (LPS) in gel-diffusion tests.Sections of Flexibacter BH3 gliding on a gold film overlaying an agar medium reveraled a highly convuluted cell envelope outer membrane, portions of which closely conformed to the microcontours of the gold surface. Possible mechanisms of gliding are discussed in relation to this close association with solid surface features, to the finding that flexibility and spiral motion are not essential for gliding, and to evidence revealing the extrusion of slime in advance of pathfinder bacteria.Abbreviations used KDO 2-keto-3-deoxyoctonate - LPS lipopolysaccharide     

Transglutaminase catalyzed incorporation of putrescine into surface proteins of mouse neuroblastoma cells

Summary Transglutaminase, purified from guinea pig liver, was used to catalyze the incorporation of [¹⁴C]putrescine into exposed surface proteins of intact mouse neuroblastoma cells. This method specifically labeled two surface proteins (Mr = 92 000 and 76 000) in the N-18 mouse neuroblastoma cells and three surface proteins (Mr = 92 000, 76 000, and 72 000) in the NB-15 mouse neuroblastoma cells. In addition, transglutaminase also catalyzed cross-linking reactions of exposed surface proteins. In both the N-18 and NB-15 cells, differentiation was accompanied by a 2-fold increase of specific radioactivity incorporated into trichloroacetic acid insoluble cellular material, suggesting that the differentiated mouse neuroblastoma cells may possess greater amount of accessible peptide-bound glutaminyl residues on their surface than their malignant counterparts. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorographic method revealed that while the [¹⁴C]putrescine-labeled protein patterns of undifferentiated and differentiated mouse neuroblastoma cells were similar, the intensity of labeling of individual bands was specifically modulated by cell differentiation.Abbreviations PMSF phenylmethylsulfonylfluoride - Bt₂cAMP,N⁶,O² Dibutyryl adenosine 3:5-cyclic monophosphate - IBMX 3-isobutyl-l-methyl xanthine - SDSPAGE sodiumdodecylsulfate-polyacrylamide gel electrophoresis - HEPES N-2-hydroxylethylpiperazine-N-2-ethanesulfonic acid     

Persistence of 14C-gibberellin A3 on the surface of Citrus fruit peel and on inert glass surfaces

The persistence of gibberellin A₃ on plant surfaces was examined using fruit of Marsh seedless grapefruit (Citrus paradisi Macf.) and an inert glass model system. ¹⁴C-gibberellin A₃ was applied to surfaces in aqueous treatment solutions or in waxing solutions. Dried-out treatment residues were removed by washing and analyzed for total and GA₃-like radioactivity. Gibberellin A₃ persisted without significant loss for at least 7 d in aqueous treatment solutions (pH 4.0 or 6.2) but was less persistent in the pH 10.4 waxing solution (t1/2=7 d).Loss of total peel surface radioactivity was fast during the first 3 days, slowing down afterwards. After 14 days 73% of the initial radioactivity could still be recovered from fruit peel surface and 70% of the recovered radioactivity was still in the form of gibberellin A₃. Gibberellin A₃ was somewhat more persistent in residues from pH 4 than pH 7 treatment solutions. Light had a slight enhancing effect on gibberellin A₃ decomposition on fruit peel under growth chamber conditions. After 12 d at 100% relative humidity, 88% of the radioactivity on glass surfaces was still in the form of gibberellin A₃, as against 45% at a relative humidity of 50%. Simulated field conditions, combining daily fluctuations in light, temperature and relative humidity, markedly enhanced gibberellin A₃ decomposition on glass surfaces (t1/2=2 d). Gibberellin A₃ was very persistent (90% after 9 d) in the waxing residues on fruit peel surface.Abbreviations GA₃ gibberellin A₃ - RH relative humidity     

In vivo distribution of cryogenically preserved guinea pig granulocytes

Granulocytes were isolated from whole blood of guinea pigs by counterflow centrifugation and labeled with [¹⁴C]diisopropylfluorophosphate ([¹⁴C]DFP). One-half of the labeled cells was injected intravenously via the femoral vein into a guinea pig, while the other half was cryogenically preserved with 5% dimethyl sulfoxide (DMSO), 6% hydroxyethyl starch (HES), and 4% human albumin, at a rate of 4 °C per minute by storage at ?80 °C and then stored for 3 days at ?80 °C. Ninety percent of the isolated granulocytes were recovered after cryogenic preservation, thawing, and washing. Aliquots before injection all produced fluorescein from fluorescein diacetate and excluded ethidium bromide. Latex ingestion was 78% and yeast ingestion was 75%. The frozen-thawed-washed-resuspended labeled granulocytes were injected into a second guinea pig. Paired animals sacrificed 35 min after injection were examined in whole-body sections for distribution of radiolabeled granulocytes to the tissues. In two pairs of animals, activity was found in the lung, liver, spleen, and kidney. The technique does not permit a distinction between fresh and cryopreserved granulocytes although there was a greater deposition of fresh cells in the liver and spleen. No activity was found in the blood of the vena cava in animals with either fresh or frozen cells. An animal injected with free [¹⁴C]DFP revealed a vascular distribution with high activity in blood, lung, and kidney, and less activity in the liver and spleen. The data indicate that radiolabeled, cryogenically preserved guinea pig granulocytes exhibited a tissue distribution qualitatively similar to fresh granulocytes, and free [¹⁴C]DFP infused without granulocytes differed qualitatively and quantitatively from fresh and cryopreserved granulocytes.     

Guinea pig model for evaluating the potential public health risk of swine and avian influenza viruses

Background

The influenza viruses circulating in animals sporadically transmit to humans and pose pandemic threats. Animal models to evaluate the potential public health risk potential of these viruses are needed.

Methodology/Principal Findings

We investigated the guinea pig as a mammalian model for the study of the replication and transmission characteristics of selected swine H1N1, H1N2, H3N2 and avian H9N2 influenza viruses, compared to those of pandemic (H1N1) 2009 and seasonal human H1N1, H3N2 influenza viruses. The swine and avian influenza viruses investigated were restricted to the respiratory system of guinea pigs and shed at high titers in nasal tracts without prior adaptation, similar to human strains. None of the swine and avian influenza viruses showed transmissibility among guinea pigs; in contrast, pandemic (H1N1) 2009 virus transmitted from infected guinea pigs to all animals and seasonal human influenza viruses could also horizontally transmit in guinea pigs. The analysis of the receptor distribution in the guinea pig respiratory tissues by lectin histochemistry indicated that both SAα2,3-Gal and SAα2,6-Gal receptors widely presented in the nasal tract and the trachea, while SAα2,3-Gal receptor was the main receptor in the lung.

Conclusions/Significance

We propose that the guinea pig could serve as a useful mammalian model to evaluate the potential public health threat of swine and avian influenza viruses.     

Functional and morphological characterization of cultures of Kupffer cells and liver endothelial cells prepared by means of density separation in Percoll,and selective substrate adherence

Summary This paper presents a study on the structure and function of Kupffer cells (KC) and liver endothelial cells (LEC) isolated by a simple and rapid technique involving 1) perfusion of the liver with collagenase; 2) cell separation by means of density centrifugation in Percoll; and 3) cell culture, taking advantage of the fact that KC and LEC differ in their preferences for growth substrate. The KC, which attach and spread under serum-free conditions on surfaces of glass or plastic during the first 15 min in culture exhibit a typical macrophage-like morphology including membrane ruffling and a heterogenous content of vacuoles. Moreover, these cells express (a) Fc receptors (FcR) for binding and phagocytosis of erythrocytes covered with immune globulin G (E-IgG), and (b) complement receptors (CR) for binding and serum dependent phagocytosis of erythrocytes covered with either human C3b or mouse inactivated C3b (iC3b). The cells also bind fluid phase fluoresceinated C3b. Approximately 30% of the KC express immune response-associated (Ia)-antigens.The LEC attach and spread on fibronectin coated surfaces, but not on glass or plastic surfaces, during the first two hours in culture with or without serum, and are morphologically distinct from KC. Cultured LEC are well spread out with no membrane ruffling and with numerous large vesicles surrounding the regularly shaped nucleus. These cells bind, but do not ingest E-IgG via the FcR, but no binding of fluid phase C3b or particle fixed C3b or iC3b can be observed. Incubation of LEC with fluorescein amine conjugates of ovalbumin or formaldehyde treated serum albumin, but not with fluoresceinated native serum albumin, results in accumulation of fluorescence specifically localized in the large perinuclear vesicles. Neither KC nor any other cell types tested have the ability to accumulate fluorescence upon incubation with these compounds. Iaantigens are not present on the LEC.Cytochemical demonstration of unspecific esterase, acid phosphatase, and peroxidase reveals different patterns and intensities of staining in KC as compared to LEC.Abbreviations Used KC Kupffer cells - LEC Liver endothelial cells - C Complement - C3b Major fragment of C3 activation - iC3b C3b that has been cleaved by factor I (C3b inactivator), present in serum - meC3b C3b produced by treating purified human C3 with methyl amine - trC3b C3b produced by treating purified human C3 with trypsin - CR Complement receptors for C3b and iC3b - IgG Immune globulin G - IgM Immune globulin M - E Erythrocytes - E-IgG E covered with anti-E IgG - E-IgM E covered with anti-E IgM - E-C3b(h) E-IgM reacted with purified human C1, C4, oxidized C2 and C3 (E-IgMC14^xyC2C3b) - E-iC3b(m) E-IgM incubated with C5 deficient serum from AKR mice - FcR Receptors for the Fc portion of IgG - FITC Fluorescein isothiocyanate - FITC-meC3b FITC conjugated to meC3b - FITC-trC3b FITC conjugated to trC3b - FA Fluorescein amine - FA-OA Ovalbumin conjugated with FA - FA-SA Serum albumin conjugated with FA - FA-FSA Formaldehyde-treated serum albumin conjugated with FA - Ia Immune response-associated AcE Acid unspecific esterase acting on alpha naphtyl acetate - NASDAE Unspecific esterase acting on naphthol AS-D acetate - NASDCAE Unspecific esterase acting on napthol AS-D chloroacetate     

The occurrence of fimbriae on a N 2 2 -fixing cyanobacterium which occurs in lichen symbiosis

The Nostoc cyanobiont of the lichen Peltigera canina when grown on N₂ possesses, in the motile stage, discrete unbranched non-flagellar appendages (fimbriae or pili). These arise from the host cell surface in a peritrichous manner, have an axial hole, are 7.0 ±0.3 nm in diameter and are up to 3 m long. They do not haemagglutinate guinea pig red blood corpuscles and differ from the major fimbrial types reported for Gram-negative heterotrophic bacteria and from sex pili. They may be involved in motility and specificity in symbiotic cyanobacteria.     

Secondary prevention of allergic symptoms in a dairy farmer by use of a milking robot

Background

Allergic sensitization and reactions to guinea pig (Cavia porcellus) have been well documented in laboratory animal handlers, primarily manifesting as rhinitis, conjunctivitis, and asthma. Severe allergic reactions, however, are rare.

Methods

We report two patients with severe allergic reactions following non-occupational exposure to guinea pigs. The first patient, an 11-year-old female, developed ocular, nasal, skin and laryngeal edema symptoms immediately after handling a guinea pig. The second patient, a 24-year-old female, developed symptoms of isolated laryngeal edema after cleaning a guinea pig cage. Percutaneous skin testing, RAST, ELISA and ELISA inhibition testing with guinea pig extract were performed.

Results

Both patients had IgE-mediated allergy to guinea pig confirmed by ELISA and either RAST or skin testing. ELISA inhibition studies confirmed the specificity of the IgE reactivity to guinea pig.

Conclusion

Severe IgE-mediated reactions can occur following non-occupational guinea pig exposure. Physicians should be aware of this possibility.     

Damage of Guinea Pig Heart and Arteries by a Trioleate-Enriched Diet and of Cultured Cardiomyocytes by Oleic Acid

Background

Mono-unsaturated fatty acids (MUFAs) like oleic acid have been shown to cause apoptosis of cultured endothelial cells by activating protein phosphatase type 2C α and β (PP2C). The question arises whether damage of endothelial or other cells could be observed in intact animals fed with a trioleate-enriched diet.

Methodology/Principal Findings

Dunkin-Hartley guinea pigs were fed with a trioleate-enriched diet for 5 months. Advanced atherosclerotic changes of the aorta and the coronary arteries could not be seen but the arteries appeared in a pre-atherosclerotic stage of vascular remodelling. However, the weight and size of the hearts were lower than in controls and the number of apoptotic myocytes increased in the hearts of trioleate-fed animals. To confirm the idea that oleic acid may have caused this apoptosis by activation of PP2C, cultured cardiomyocytes from guinea pigs and mice were treated with various lipids. It was demonstrable that oleic acid dose-dependently caused apoptosis of cardiomyocytes from both species, yet, similar to previous experiments with cultured neurons and endothelial cells, stearic acid, elaidic acid and oleic acid methylester did not. The apoptotic effect caused by oleic acid was diminished when PP2C α and β were downregulated by siRNA showing that PP2C was causally involved in apoptosis caused by oleic acid.

Conclusions/Significance

The glycerol trioleate diet given to guinea pigs for 5 months did not cause marked atherosclerosis but clearly damaged the hearts by activating PP2C α and β. The diet used with 24% (wt/wt) glycerol trioleate is not comparable to human diets. The detrimental role of MUFAs for guinea pig heart tissue in vivo is shown for the first time. Whether it is true for humans remains to be shown.     

Histochemical comparison of oxidative enzymes in adrenal glands of mammals

Summary Succinic dehydrogenase, five DPN-linked dehydrogenases-lactic dehydrogenase, malic dehydrogenase, glutamic dehydrogenase, -glycerophosphate dehydrogenase, -hydroxybutyric dehydrogenase, two TPN linked dehydrogenases — glucose-6-phosphate dehydrogenase, isocitric dehydrogenase and 3-ol steroid dehydrogenase were studied in mouse, rat, guinea pig, rabbit, dog, cat, cow, monkey and human adrenal glands. Histochemical studies were made of a characteristic distribution of different level of enzyme activity. In mammals adrenal glands, glucose-6-phosphate dehydrogenase showed the highest activity and its localization was divided into the following two groups: 1) High enzymatic activity was demonstrated in the zona fasciculata and reticularis of the rat, guinea pig, rabbit, cat and 2) high enzymatic activity was demonstrated in the zona glomerulosa and reticularis of the dog, cow and monkey. A precise relationship between the localization and endocrinological function remains abscure.     

Killing ofLegionella pneumophila by human serum and iron-binding agents

The inhibitory effect of serum on the growth and survival ofLegionella pneumophila Bloomington 2 was investigated. When incubated in the presence of 20%–50% normal human serum for 10 h, viability was decreased by >99%. Heat-inactivated or <40% normal serum supplemented with 50 M iron was not inhibitory. The addition of guinea pig complement to heat-inactivated serum resulted in killing of approximately 98% of the cells. Growth in buffered yeast extract broth was inhibited by the addition of ferric iron-binding compounds. Minimum bactericidal concentrations at 37°C were 10 M apotransferrin, 35 M 1,10-phenanthroline, and 50 M deferoxamine. Addition of iron chelators to normal serum did not accelerate killing. Egg yolk-passaged virulent strains and agar-grown avirulent strains exhibited similar serum sensitivity. Results of this study indicate that complement and serum transferrin are antagonistic to the growth ofLegionella in serum.     

Structure of the Altitude Adapted Hemoglobin of Guinea Pig in the R2-State

Background

Guinea pigs are considered to be genetically adapted to a high altitude environment based on the consistent finding of a high oxygen affinity of their blood.

Methodology/Principal Findings

The crystal structure of guinea pig hemoglobin at 1.8 Å resolution suggests that the increased oxygen affinity of guinea pig hemoglobin can be explained by two factors, namely a decreased stability of the T-state and an increased stability of the R2-state. The destabilization of the T-state can be related to the substitution of a highly conserved proline (P44) to histidine (H44) in the α-subunit, which causes a steric hindrance with H97 of the β-subunit in the switch region. The stabilization of the R2-state is caused by two additional salt bridges at the β1/β2 interface.

Conclusions/Significance

Both factors together are supposed to serve to shift the equilibrium between the conformational states towards the high affinity relaxed states resulting in an increased oxygen affinity.     

Isolation and characterization of a Forssman antigen-binding lectin from velvet bean (Mucuna derringiana) seeds

A Forssman antigen (GalNAc1-3GalNAc1-3Gal1-4Gal1-4Glc1-1Cer)-binding lectin has been purified from velvet bean (Mucuna derringiana) seeds by a combination of affinity chromatography and reversed phase HPLC. This lectin agglutinates both native and trypsin-treated sheep erythrocytes as well as trypsinized rabbit erythrocytes, but neither native rabbit nor human erythrocytes, irrespective of blood group type. SDS-PAGE and gel filtration chromatography reveal the lectin to be a homodimer consisting of two 54 kDa subunits linked by non-covalent bonds. The results obtained by quantitative precipitation, haemagglutination inhibition and TLC overlay assays indicate that theMucuna lectin specifically recognizes Forssman antigen and Forssman disaccharide (GalNAc1-3GalNAc)-related structures. Abbreviations: The abbreviations and the trivial names used are: AH, 6-aminohexyl; BSA, bovine serum albumin; Cer, ceramide; HPLC, high performance liquid chromatography; PAGE, polyacrylamide gel electrophoresis; PBS, 10mm phosphate-buffered saline, pH 7,2, containing 0.15m NaCl; PMSF, phenyl methyl sulfonyl fluoride; SDS, sodium dodecyl sulphate; TFA, trifluoroacetic acid; TBS, 20mm tris-buffered saline, pH 7.2; TLC, thin-layer chromatography; A disaccharide, GalNAc1-3Gal; A trisaccharide, GalNAc1-3[Fuc1-2]Gal; Forssman disaccharide, GalNAc1-3GalNAc; CDH (ceramide dihexoside or lactosyl ceramide) Gal1-4Glc1-1Cer (LacCer); CTH (ceramide trihexoside or globotriosyl ceramide), Gal1-4Gal1-4Glc1-1Cer (GbOse₃Cer or Gb₃); globoside (globotetraosyl ceramide), GalNAc1-3Gal1-4Gal1-4Glc1-1Cer (GbOse₄Cer or Gb₄); Forssman antigen (globopentaosyl ceramide), GalNAc1-3GalNAc1-3Gal1-4Gal1-4Glc1-1Cer (GbOse₅Cer).     

Inhibition of an vitro cell-mediated response: further experiments on the cell lineage and target of suppressor cells

The contribution of lymphotoxin to guinea pig leukocyte natural cytotoxicity was evaluated with [³H]TdR release and colony-inhibition assays of 104C1 benzo(a)pyrene in vitro-transformed and tumorigenic, tumor-specific transplantation antigen-negative, syngeneic strain 2/ N fibroblasts. Cytolethal ³H-release activities of mitogen (PHA)¹-stimulated nonimmune and ovalbumin (OA) immune as well as OA-stimulated OA immune unfractionated, adherent (macrophage-enriched) and nonadherent peritoneal leukocytes are qualitatively similar. ³H release is maximal by 48 hr, increases with antigen or mitogen concentration, is greatest with unfractionated leukocytes, and is least with adherent macrophages. Lymphotoxin produced by peritoneal leukocytes, alone or in combination with the leukocytes does not or only minimally induces ³H release even after 6 days of incubation with guinea pig target cells although guinea pig lymphotoxin possesses cytolytic activity as indicated by ³H release from αL929 mouse tumor cells. In contrast to the absent or very weak cytolytic activity of guinea pig lymphotoxin for the guinea pig target cells nonimmune macrophages, nonadherent leukocytes, and lymphotoxin all exhibit readily detectable colony-inhibitory (CI) activity for the syngeneic tumor cells. Macrophage and lymphotoxin CI, moreover, are additive, whereas nonadherent leukocyte and lymphotoxin CI are synergistic. The latter may be due to additional lymphotoxin induced by target cell antigens or other mechanisms of target cell stimulation of effector lymphoid cells and result from very high local levels of lymphotoxin released by the effector cells. Lymphotoxin CI, furthermore, can be cytostatic or cytolethal as indicated by resumption of 104C1 but not αL929 colony growth following removal of lymphotoxin, indicating that natural cell-mediated cytotoxicity consists of lymphotoxin-dependent and -independent cytostatic and cytolethal effector mechanisms.     

Cloning and expression of the A2a adenosine receptor from guinea pig brain

A full-length complementary DNA (cDNA) clone encoding the guinea pig brain A₂ adenosine receptor has been isolated by polymerase chain reaction (PCR) and low-stringency-hybridization screening of a guinea pig brain cDNA library. This cDNA contains a long open reading frame encoding a 409-amino acid-residue protein which is highly homologous to the A₂ adenosine receptors previously cloned from other species. Hydrophobicity analysis of the deduced protein sequence reveals seven hydrophobic regions, characteristic of a member of the G-protein-coupled receptor superfamily. Radioligand binding assay and functional (GTPase and cAMP) assays of the receptor, transiently expressed in mammalian cells, demonstrate typical characteristics of the A₂ type adenosine receptor. The messenger RNA (mRNA) of this A₂ receptor is found in the brain, heart, kidney and spleen. Receptor autoradiography with [³H]CGS21680, a specific A₂ agonist, and in situ hybridization with A₂ cRNA probe in guinea pig brain indicate that the receptor is expressed exclusively in the caudate nucleus. The pharmacological profile and anatomical distribution of this receptor indicate that it is of the A_2a subtype. This work represents the first cloning of an A_2a receptor in a rodent species, offers a complete pharmacological characterization of the receptor and provides an anatomical comparison between binding profile and gene expression of the receptor.Abbreviations ADAC adenosine amine congener - BA N⁶-benzyladenosine - bp nucleotide base pair - cAMP cyclic adenosine 3,5-monophosphate - CCPA 2-chloro-N⁶-cyclopentyladenosine - CGS 21680 2-p-(2-carboxyethyl)phenethylamino-5-N-ethylcarboxamido adenosine hydrochloride - CHA N⁶-cyclohexyladenosine - CNS central nervous system - CPA N⁶-cyclohexyladenosine - CNS central nervous system - CPA N⁶-cyclopentyladenosine - CPX 8-cyclopentyl-1,3-dipropylxanthine - DME Dulbecco's modified Eagle's medium - DMPX 3,7-dimethyl-1-propargylxanthine - DPMX 1,3-dipropyl-7-methylxanthine - DPX 1,3-dipropyl-8-(2-amono-4-chlorophenyl)xanthine - FCS fetal calf serum - IBMX 3-isobutyl-1-methylxanthine - KHB Kreb-HEPES buffer - MECA 5-N-methylcarboxamidoadenosine - NECA 5-N-ethylcarboxamidoadenosine - D-PBS Dulbecco's phosphate buffered saline - PCR polymerase chain reaction - R-PIA R(–)-N⁶-(2-phenylisopropyl)adenosine - SSPE sodium chloride-sodium phosphate-EDTA buffer - TM transmembrane domain - XAC xanthine amine congener Special issue dedicated to Dr. Bernard W. Agranoff     

Response of net ecosystem gas exchange to a simulated precipitation pulse in a semi-arid grassland: the role of native versus non-native grasses and soil texture

Physiological activity and structural dynamics in arid and semi-arid ecosystems are driven by discrete inputs or pulses of growing season precipitation. Here we describe the short-term dynamics of ecosystem physiology in experimental stands of native (Heteropogon contortus) and invasive (Eragrostis lehmanniana) grasses to an irrigation pulse across two geomorphic surfaces with distinctly different soils: a Pleistocene-aged surface with high clay content in a strongly horizonated soil, and a Holocene-aged surface with low clay content in homogenously structured soils. We evaluated whole-ecosystem and leaf-level CO₂ and H₂O exchange, soil CO₂ efflux, along with plant and soil water status to understand potential constraints on whole-ecosystem carbon exchange during the initiation of the summer monsoon season. Prior to the irrigation pulse, both invasive and native grasses had less negative pre-dawn water potentials ( _pd), greater leaf photosynthetic rates (A _net) and stomatal conductance (g _s), and greater rates of net ecosystem carbon exchange (NEE) on the Pleistocene surface than on the Holocene. Twenty-four hours following the experimental application of a 39 mm irrigation pulse, soil CO₂ efflux increased leading to all plots losing CO₂ to the atmosphere over the course of a day. Invasive species stands had greater evapotranspiration rates (ET) immediately following the precipitation pulse than did native stands, while maximum instantaneous NEE increased for both species and surfaces at roughly the same rate. The differential ET patterns through time were correlated with an earlier decline in NEE in the invasive species as compared to the native species plots. Plots with invasive species accumulated between 5% and 33% of the carbon that plots with the native species accumulated over the 15-day pulse period. Taken together, these results indicate that system CO₂ efflux (both the physical displacement of soil CO₂ by water along with plant and microbial respiration) strongly controls whole-ecosystem carbon exchange during precipitation pulses. Since CO₂ and H₂O loss to the atmosphere was partially driven by species effects on soil microclimate, understanding the mechanistic relationships between the soil characteristics, plant ecophysiological responses, and canopy structural dynamics will be important for understanding the effects of shifting precipitation and vegetation patterns in semi-arid environments.     

Regulation of myosin sliding alongChara actin bundles by native skeletal muscle tropomyosin

Summary Native tropomyosin from rabbit skeletal muscle introduced by intracellular perfusion intoChara cells inhibited the cytoplasmic streaming irrespective of the Ca²⁺ concentration. To find the action site of native tropomyosin inChara, the cytoplasmic streaming was reconstituted by introducing isolated endoplasm into actin donorChara cells from which native endoplasm had been removed. The reconstituted streaming was inhibited by pretreatment of the actin donor cells with native tropomyosin but not by that of the endoplasm, suggesting that the native tropomyosin inhibited the cytoplasmic streaming by binding toChara actin bundles. Staining of the actin bundles with FITC-labeled native tropomyosin also showed that the native tropomyosin could bind to the actin bundles. Streaming reconstituted fromChara actin bundles and skeletal muscle myosin was insensitive to Ca²⁺, but became sensitive on application of the native tropomyosin.Abbrevations APW artificial pond water - ATP adenosine 5-triphosphoric acid - BSA bovine serum albumin - EDTA ethylene diamine tetraacetic acid - EGTA ethyleneglycol-bis-(-aminoethylether) N,N,N,N-tetraacetic acid - FITC fluorescein isothiocyanate - FITC-NTM fluorescein isothiocyanate-labeled native tropomyosin - NTM native tropomyosin