An autoantibody against N(ε)-(carboxyethyl)lysine (CEL): possible involvement in the removal of CEL-modified proteins by macrophages

Advanced glycation end products (AGEs) are believed to play a significant role in the development of diabetic complications. In this study, we measured the levels of autoantibodies against several AGE structures in healthy human plasma and investigated the physiological role of the autoantibodies. A high titer of the autoantibody against N(ε)-(carboxyethyl)lysine (CEL) was detected in human plasma compared with other AGE structures such as CML and pentosidine. The purified human anti-CEL autoantibody reacted with CEL-modified human serum albumin (CEL-HSA), but not CML-HSA. A rabbit polyclonal anti-CEL antibody, used as a model autoantibody against CEL, accelerated the uptake of CEL-HSA by macrophages, but did not enhance the uptake of native HSA. Furthermore, when (125)I-labeled CEL-HSA was injected into the tail vein of mice, accumulation of (125)I-CEL-HSA in the liver was accelerated by co-injection of the rabbit anti-CEL antibody. These results demonstrate that the autoantibody against CEL in plasma may play a role in the macrophage uptake of CEL-modified proteins.     

Immunoreactivity of subunits of the (Na + K)-ATPase Cross-reactivity of the α,α+ and β forms in different organs and species

The immunologic cross-reactivity of the α and α+ forms of the large subunit and the β subunit of the (Na⁺ + K⁺)-ATPase from brain and kidney preparations was examined using rabbit antiserum prepared against the purified holo lamb kidney enzyme. As previously reported by Sweadner ((1979) J. Biol. Chem. 254, 6060–6067) phosphorylation of the large subunit of the (Na⁺ + K⁺)-ATPase in the presence of Na⁺, Mg²⁺, and [γ-³²P]ATP revealed that dog and, very likely, rat brain contain two forms of the large subunit (designated α and α+) while dog, rat, and lamb kidney contain only one form (α). The cross-reactivity of the α and α+ forms in these preparations was investigated by resolving the subunits by SDS-polyacrylamide gel electrophoresis. The separated polypeptides were transferred to unmodified nitrocellulose paper, and reacted with rabbit anti-lamb kidney serum, followed by detection of the antigen-antibody complex with ¹²⁵I-labeled protein A and autoradiography. By this method, the α and α+ forms of rat and dog brain, as well as the α form found in kidney, were shown to cross-react. In addition, membranes from human cerebral cortex were shown to contain two immunoreactive bands corresponding to the α and α+ forms of dog brain. In contrast, the brain of the insect Manduca sexta contains only one immunoreactive polypeptide with a molecular weight intermediate to the α and α+ forms of dog brain. The β subunit from lamb, dog and rat kidney and from dog and rat brain cross-reacts with anti-lamb kidney (Na⁺ + K⁺)-ATPase serum. The mobility of the β subunit from dog and rat brain on SDS-polyacrylamide electrophoresis gels is greater than the mobility of the β subunit from lamb, rat or dog kidney.     

Isolation and characterization of monoclonal antibodies to (Na + K)-ATPase

Four stable hybridoma cell lines secreting antibodies specific to the membrane (Na⁺ + K⁺)-dependent ATPase isolated from lamb kidney medulla have been produced by fusing mouse myeloma cells with spleen cells from immunized mice. These cell lines produce IgG γ₁ heavy chain and κ light chain antibodies which are directed against the catalytic or α-subunit of the (Na⁺ + K⁺)-ATPase enzyme. Binding studies, using antibodies that were produced by growing hybridomas in vivo and purified by affinity column chromatography, suggest a somewhat higher affinity of these antibodies for the isolated α-subunit than for the ‘native’ holoenzyme. In addition, these monoclonal antibodies show no reactivity with either the glycoprotein (β) subunit of the lamb enzyme nor the (Na⁺ + K⁺)-ATPase from rat kidney, an ouabain-insensitive organ. Cotitration binding experiments have shown that the antibodies from two cell lines originally isolated independently from the same culture plate well population of fused cells bind to the same determinant site and are probably the same antibody. Cotitration and competition binding studies with two other antibodies have revealed two additional distinct antibody binding sites which appear to have little overlap with the first site. One of the three different antibodies isolated caused a partial inhibition of the (Na⁺ + K⁺)-ATPase activity. This antibody appears to be directed against a specific functionally important site of the α-subunit and is a competitive inhibitor of ATP binding. Under optimum conditions of ATPase activity, this inhibitory effect is not altered by the presence of the other two antibodies.     

Analysis of a Vesicular Glutamate Transporter (VGLUT2) Supports a Cell-leakage Mode in Addition to Vesicular Packaging

VGLUT2 is one of three vesicular glutamate transporters that play crucial roles in glutamatergic excitatory neurotransmission. We explored the functional properties of the rat VGLUT2 by heterologous expression of VGLUT2 in Xenopus oocytes. Immunocytochemical analysis indicated that most VGLUT2 protein was expressed in intracellular compartments but that some expression occurred also on the plasma membrane. Functional analysis revealed VGLUT2 to be active in two independent modes, namely, uptake into intracellular organelles and efflux at the plasma membrane. VGLUT-specific transport was identified based on the strong preference for glutamate over aspartate—in contrast to plasma-membrane or mitochondrial glutamate transporters—and sensitivity to known VGLUT blockers. VGLUT2 expression in oocytes (1) stimulated the influx of l-[³H]glutamate, but not d-[³H]aspartate, into digitonin-permeabilized oocytes and (2) stimulated efflux of l-glutamate, but not l-aspartate, from intact oocytes preinjected with ³H-labeled amino acids. In the latter assay, cellular efflux of glutamate (which was blocked by rose bengal and trypan blue) may be analogous to vesicular packaging of glutamate. Our data are consistent with VGLUT2-mediated H⁺/l-glutamate antiport, but not antiport with chloride. Expression of mammalian VGLUT1 and VGLUT3 also stimulated l-[³H]glutamate efflux from Xenopus oocytes, suggesting that this phenomenon is a general feature of vesicular glutamate transporters. Our findings support the idea that vesicular glutamate transporters, when transiently expressed on the neuronal plasma membrane, may mediate Ca²⁺-independent glutamate leakage in addition to their traditional role of packaging glutamate into synaptic vesicles for Ca²⁺-dependent exocytosis. Special issue article in honor of Dr. Frode Fonnum.     

Nitrogen Transfer Within and Between Plants Through Common Mycorrhizal Networks (CMNs)

Mycorrhizae play a critical role in nutrient capture from soils. Arbuscular mycorrhizae (AM) and ectomycorrhizae (EM) are the most important mycorrhizae in agricultural and natural ecosystems. AM and EM fungi use inorganic NH₄ ⁺ and NO₃ ^?, and most EM fungi are capable of using organic nitrogen. The heavier stable isotope ¹⁵N is discriminated against during biogeochemical and biochemical processes. Differences in ¹⁵N (atom%) or δ¹⁵N (‰) provide nitrogen movement information in an experimental system. A range of 20 to 50% of one-way N-transfer has been observed from legumes to nonlegumes. Mycorrhizal fungal mycelia can extend from one plant's roots to another plant's roots to form common mycorrhizal networks (CMNs). Individual species, genera, even families of plants can be interconnected by CMNs. They are capable of facilitating nutrient uptake and flux. Nutrients such as carbon, nitrogen and phosphorus and other elements may then move via either AM or EM networks from plant to plant. Both ¹⁵N labeling and ¹⁵N natural abundance techniques have been employed to trace N movement between plants interconnected by AM or EM networks. Fine mesh (25～45 μm) has been used to separate root systems and allow only hyphal penetration and linkages but no root contact between plants. In many studies, nitrogen from N₂-fixing mycorrhizal plants transferred to non-N₂–fixing mycorrhizal plants (one-way N-transfer). In a few studies, N is also transferred from non-N₂–fixing mycorrhizal plants to N₂-fixing mycorrhizal plants (two-way N-transfer). There is controversy about whether N-transfer is direct through CMNs, or indirect through the soil. The lack of convincing data underlines the need for creative, careful experimental manipulations. Nitrogen is crucial to productivity in most terrestrial ecosystems, and there are potential benefits of management in soil-plant systems to enhance N-transfer. Thus, two-way N-transfer warrants further investigation with many species and under field conditions.     

Calcium (45Ca) accumulation and transport in chicory (Cichorium intybus L.) root during bud development (forcing)

The lower part (4 cm) of the witloof chicory tap-root (15 cm) was immersed in a complete nutrient solution for 21 days, in the darkness at 18°C and at high RH. This process of forcing which leads to the emergence of an etiolated bud (chicon) was associated with a decrease in root dry weight. Although the amount of calcium in the root and the root cationic exchange capacity remained constant during forcing, the net uptake of calcium, negligible at the onset of forcing, progressively increased to a rate after ten days of 45 mol day^–1. Absorption of ⁴⁵Ca remained at a constant high rate, while the initially low upward migration of ⁴⁵Ca within the root and the chicon accelerated markedly. This upward migration was associated with a progressive decline in the release of newly absorbed ⁴⁵Ca. The data support the hypothesis that calcium acquisition by witloof chicory root is predominantly determined by calcium efflux. As the forcing progressed, the influx remained almost constant while a large decrease in the efflux led to a net uptake of calcium. Upward translocation was probably linked to the formation of new negative exchange sites within the growing chicon. The hypothesis that calcium movement occurred along a preferential pathway (xylem vessels) or involved a mass movement through the root is discussed.     

Human and murine anti-DNA antibodies induce the production of anti-idiotypic antibodies with autoantigen-binding properties (epibodies) through immune-network interactions

To examine the potential role of immune-network interactions in the production of lupus autoantibodies, normal NZW rabbit antibody responses were analyzed after immunization with one of the following Ig preparations: human lupus serum anti-dsDNA antibodies, human lupus serum anti-ssDNA antibodies, a mixture of human lupus serum anti-dsDNA and anti-ssDNA antibodies, the MRL-lpr/lpr anti-dsDNA mAb H241, and the MRL-lpr/lpr anti-ssDNA mAb H130. Four of five rabbits produced Ig typical of lupus autoantibodies: individual rabbit Ig cross-reacted with multiple autoantigens including nucleic acids, cardiolipin, SmRNP, glomerular extract, laminin, and exogenous Ag. Rabbit anti-Id against human anti-dsDNA antibodies were highly specific for dsDNA. Notably, in each serum the autoantibody activity was confined to the anti-Id Ig fraction. A similar spontaneously occurring Id-anti-Id interaction was also found between anti-ssDNA and anti-dsDNA antibodies isolated from an individual lupus patient. These results indicate that lupus autoantibodies which share Ag binding properties with pathogenic Ig, including both cross-reactive and anti-dsDNA antibodies, can induce the production of Ig with similar autoantigen binding properties through immune-network interactions. This phenomenon, if unregulated, could lead to the amplification of pathogenic autoantibody production in individuals with systemic lupus.     

Profiling humoral autoimmune repertoire of dilated cardiomyopathy (DCM) patients and development of a disease-associated protein chip

Dilated cardiomyopathy (DCM) is a myocardial disease characterized by progressive depression of myocardial contractile function and ventricular dilatation. Thirty percent of DCM patients belong to the inherited genetic form; the rest may be idiopathic, viral, autoimmune, or immune-mediated associated with a viral infection. Disturbances in humoral and cellular immunity have been described in cases of myocarditis and DCM. A number of autoantibodies against cardiac cell proteins have been identified in DCM. In this study, we have profiled the autoantibody repertoire of plasma from DCM patients against a human protein array consisting of 37,200 redundant, recombinant human proteins and performed qualitative and quantitative validation of these putative autoantigens on protein microarrays to identify novel putative DCM specific autoantigens. In addition to analyzing the whole IgG autoantibody repertoire, we have also analyzed the IgG3 antibody repertoire in the plasma samples to study the characteristics of IgG3 subclass antibodies. By combining screening of a protein expression library with protein microarray technology, we have detected 26 proteins identified by the IgG antibody repertoire and 6 proteins bound by the IgG3 subclass. Several of these autoantibodies found in plasma of DCM patients, such as the autoantibody against the Kv channel-interacting protein, are associated with heart failure.     

Involvement of polyamines in the inhibiting effect of injury caused by cutting on K+ uptake through the plasma membrane

The inhibition of K⁺ uptake through the plasma membrane resulting from injury caused by cutting, or from application of polyamines (PAs), has been investigated in root segments of maize (Zea mays L.) and pea (Pisum sativum L.). It was found, for both treatments, that K⁺ uptake recovered if the segments were washed for 2 h. The K⁺ uptake inhibited by cutting and that inhibited by spermidine treatment were stimulated to the same extent by fusicoccin. In addition, there was a correlation between the extent of the recovery of K⁺ uptake caused by washing and the distribution, along the root axis, of both PAs and the activities of enzymes responsible for PA degradation. In apical segments of maize, where the PA content and the activity of the degradative enzyme polyamine oxidase (EC 1.5.3.3) were higher than in the more distal segments, the recovery of K⁺ uptake caused by washing was also higher. On the other hand, the opposite trend was observed in root segments of pea, where the PA content and the activity of the degradative enzyme diamine oxidase (EC 1.4.3.6) were higher in distal segments in which K⁺ uptake was greatly stimulated by washing. The effect of the amine-oxidase inhibitor, aminoguanidine, indicates that the degradation products of PAs are involved in the mechanism of inhibition of K⁺ uptake by PAs. The data also seem to indicate that PAs and their degradation products are responsible for the inhibition of K⁺ uptake occurring as a result of injury sustained by cutting roots into segments.Abbreviations DAO diamine oxidase - FC fusicoccin - PA polyamine - PAO polyamine oxidase - PUT putrescine - SPD spermidine     

Chum salmon (Oncorhynchus keta) stanniocalcin inhibitis in vitro intestinal calcium uptake in Atlantic cod (Gadus morhua)

Summary Chum salmon (Oncorhynchus keta) stanniocalcin was purified, partially identified and tested for bioactivity in an assay on the intestinal calcium uptake in a marine teleost (Gadus morhua). Basic ethanol extraction, ion exchange chromatography, gel filtration and reverse-phase high-performance liquid chromatography resulted in the isolation of a homogenous glycoprotein that appears as a 46-kDa product under non-reducing conditions and as a 23-kDa product under reducing conditions after sodium dodecylsulphate-polyacrylamide gel electrophoresis. The glycoprotein is likely to be a homodimer composed of two subunits of 23 kDa each. Further characterization indicates homology to Australian eel, sockeye salmon, coho salmon and rainbow trout stanniocalcin, and the glycoprotein is thus concluded to be stanniocalcin. Stanniocalcin-like immunoreactivity was demonstrated in the corpuscles of Stannius of the Atlantic cod, with a specific antiserum raised against purified chum salmon stanniocalcin. The physiological importance and the biological activity of chum salmon stanniocalcin was tested by evaluating its effect on intestinal calcium uptake by the Atlantic cod in vitro. The intestine was perfused, both vascularly and through the intestinal lumen, and the calcium mucosa-to-serosa flux was measured using ⁴⁵Ca²⁺ as a tracer. Stanniocalcin decreased the intestinal calcium uptake in a dose-related manner by 13.5% and 22.4% at doses of 2.2 and 10.9 nM stanniocalcin, respectively. The results establish the intestine as a target organ for stanniocalcin in marine teleosts.Abbreviations BIS balanced intestinal solution - CS corpuscles of Stannius - dpm disintegrations per minute - FW freshwater - J _in ^Ca influx of calcium across the intestinal mucosa - MW molecular weight - NRS normal rabbit serum - PBS phosphate buffered saline - PBST phosphate buffered saline containing 0.05% Tween-20 - PITC phenyl isothiocyanate - rp-HPLC reverse phase - SW seawater - STC phenyl isothiocyanate - rp-HPLC reverse phase - SW seawater - STC stanniocalcin - TFA Trifluoroacetic acid - Tris Tris(hydroxymethyl) aminomethan - V volume per fraction     

Na+-transporting ATPase in the plasma membrane of halotolerant microalga Dunaliella maritima operates as a Na+ uniporter

A fraction of inside-out membrane vesicles enriched in plasma membranes (PM) was isolated from Dunaliella maritima cells. Attempts were made to reveal ATP-driven Na⁺-dependent H⁺ efflux from the PM vesicles to external medium, as detected by alkalization of the vesicle lumen. In parallel experiments, ATP-dependent Na⁺ uptake and electric potential generation in PM vesicles were investigated. The alkalization of the vesicle lumen was monitored with an impermeant pH-sensitive optical probe pyranine (8-hydroxy-1,3,6-pyrenetrisulfonic acid), which was loaded into vesicles during the isolation procedure. Sodium uptake was measured with ²²Na⁺ radioactive label. The generation of electric potential in PM vesicles (positive inside) was recorded with a voltage-sensitive probe oxonol VI. Appreciable Na⁺-and ATP-dependent alkalization of vesicle lumen was only observed in the presence of a protonophore CCCP (carbonyl cyanide-chlorophenylhydrazone). In parallel experiments, CCCP accelerated the ATP-dependent ²²Na⁺ uptake and abolished the electric potential generated by the Na⁺-ATPase at the vesicle membrane. A permeant anion NO^? ₃ accelerated ATP-dependent ²²Na⁺ uptake and promoted dissipation of the electric potential like CCCP did. At the same time, NO^? ₃ inhibited the ATP-and Na⁺-dependent alkalization of the vesicle lumen. The results clearly show that the ATP-and Na⁺-dependent H⁺ efflux from PM vesicles of D. maritima is driven by the electric potential generated at the vesicle membrane by the Na⁺-ATPase. Hence, the Na⁺-transporting ATPase of D. maritima carries only one ion species, i.e., Na⁺. Proton is not involved as a counter-ion in the catalytic cycle of this enzyme.     

Ca2+ pump and Ca2+/H+ antiporter in plasma membrane vesicles isolated by aqueous two-phase partitioning from corn leaves

Summary Plasma membrane vesicles, which are mostly right side-out, were isolated from corn leaves by aqueous two-phase partitioning method. Characteristics of Ca²⁺ transport were investigated after preparing inside-out vesicles by Triton X-100 treatment.⁴⁵Ca²⁺ transport was assayed by membrane filtration technique. Results showed that Ca²⁺ transport into the plasma membrane vesicles was Mg-ATP dependent. The active Ca²⁺ transport system had a high affinity for Ca²⁺(K _m (Ca²⁺)=0.4 m) and ATP(K _m (ATP)=3.9 m), and showed pH optimum at 7.5. ATP-dependent Ca²⁺ uptake in the plasma membrane vesicles was stimulated in the presence of Cl^– or NO ₃ ^– . Quenching of quinacrine fluorescence showed that these anions also induced H⁺ transport into the vesicles. The Ca²⁺ uptake stimulated by Cl^– was dependent on the activity of H⁺ transport into the vesicles. However, carbonylcyanidem-chlorophenylhydrazone (CCCP) and VO ₄ ^3– which is known to inhibit the H⁺ pump associated with the plasma membrane, canceled almost all of the Cl^–-stimulated Ca²⁺ uptake. Furthermore, artificially imposed pH gradient (acid inside) caused Ca²⁺ uptake into the vesicles. These results suggest that the Cl^–-stimulated Ca²⁺ uptake is caused by the efflux of H⁺ from the vesicles by the operation of Ca²⁺/H⁺ antiport system in the plasma membrane. In Cl^–-free medium, H⁺ transport into the vesicles scarcely occurred and the addition of CCCP caused only a slight inhibition of the active Ca²⁺ uptake into the vesicles. These results suggest that two Ca²⁺ transport systems are operating in the plasma membrane from corn leaves, i.e., one is an ATP-dependent active Ca²⁺ transport system (Ca²⁺ pump) and the other is a Ca²⁺/H⁺ antiport system. Little difference in characteristics of Ca²⁺ transport was observed between the plasma membranes isolated from etiolated and green corn leaves.     

Introduction of antibody (PL/IM 430) to a 100 kDa protein into permeabilised platelets inhibits intracellular sequestration of Ca2+

A monoclonal antibody (PL/IM 430), previously found to inhibit the uptake of Ca²⁺ into highly purified platelet intracellular membrane vesicles (Hack, N., Wilkinson, J. M. and Crawford, N. 1988,Biochem. J. 250, 355–361) has been introduced into saponin-permeabilised platelets. At a saponin concentration (20–25 g/ml) commensurate with total LDH release, sequestration of Ca²⁺ into intracellular non-mitochondrial stores is inhibited by the antibody (50% inhibition at 20 g/ml IgG). At higher saponin concentrations when intracellular binding of¹²⁵I-labelled mAb is maximum, inhibition of Ca²⁺ sequestration approaches 70%. The inhibition is specific, control studies with non-platelet directed mouse IgG and mAbs which immunoblot platelet antigens other than the 100 kDa protein did not affect the Ca²⁺ sequestration.No effect of the antibody were observed against IP₃-induced release of prestored Ca²⁺, either in permeabilised platelets or with isolated intracellular membrane vesicles. The mAb PL/IM 430 appears to bind only to the Ca²⁺ translocating channel protein associated with the intracellular membrane (Ca²⁺+Mg²⁺) ATPase and not to Ca²⁺ channels responsive to IP₃.Abbreviations mAb monoclonal antibody - PBS phosphate buffered saline - LDH lactate dehydrogenase     

Tributylbenzylammonium (TBBA+)-dependent fusicoccin (FC)-induced H+ extrusion in maize roots: relationship between the stimulating effects of TBBA+ on H+ extrusion and on Cl- efflux

The mechanism of the stimulating effect of lipophilic cations on H⁺ extrusion in maize root segments (Zea mays L.) has been investigated. The measurement of the uptake of [³H]tributylbenzylammonium ([³H]TBBA⁺), the most active lipophilic cation on H⁺ extrusion, indicated that although a relevant fraction of TBBA⁺ taken up by the tissue is adsorbed to cell surfaces, a fraction of the cation enters the cells. However no correlation was observed between the rate of TBBA⁺ uptake and that of H⁺ extrusion. On the other hand, the lipophilic cations active on H⁺ extrusion (TBBA⁺ and dibenzyldimethylammonium (DDA⁺)), in the presence of fusicoccin (FC), induced under the same conditions an efflux of Cl^-, while tetramethylammonium (TMA⁺), inactive on H⁺ extrusion, did not. The stimulation of Cl^- efflux by TBBA⁺ was independent of the anion present in the medium and was inhibited by Na-orthovanadate, an inhibitor of plasma membrane ATPase and of TBBA⁺-induced H⁺ extrusion. These results suggest that the stimulation of H⁺ extrusion by TBBA⁺ depends on its effect on Cl^- efflux rather than on its penetration into the cells.Abbreviations DDA⁺ dibenzyldimethylammonium - FC fusicoccin - 3-O-MG 3-O-methyl glucose - PD transmembrane electric potential difference - TBBA⁺ tributylbenzylammonium - TCF tissue concentration factor - TMA⁺ tetramethylammonium - TPB^- tetraphenylboron     

Bicuculline-insensitive GABA-gated Cl<Superscript>−</Superscript> channels in the larval nervous system of the moth<Emphasis Type="Italic"> Manduca sexta</Emphasis>

GABA-gated Cl^– channels were studied in the nervous system of the larval tobacco hawk moth, Manduca sexta, using electrophysiology, ³⁶Cl^– uptake into membrane microsacs and immunocytochemistry. A GABA-induced increase in Cl^– conductance was recorded from a visually identifiable neurone (fg1) in the desheathed frontal ganglion. The response was insensitive to the vertebrate GABA_A receptor antagonist, bicuculline, but was blocked by picrotoxinin. Bicuculline-insensitive, picrotoxinin-sensitive, GABA-stimulated ³⁶Cl^– uptake was also detected in membrane microsacs prepared from the isolated larval M. sexta nervous system. Such receptors appear to be the major type of GABA receptor in larval nervous system membrane microsac preparations. An antibody raised against a 17 amino acid peptide, based on the predicted C-terminus of the Drosophila GABA receptor subunit (RDL), stained not only cell bodies, including that of fg1, but also the neuropile in the frontal ganglion, indicating the existence of RDL-like GABA receptor subunits in neurones of this ganglion. Thus, bicuculline-insensitive GABA-gated Cl^– channels are present in the larval nervous system of M. sexta.     

Distribution of intravenously injected A-layer protein and lipopolysaccharide (LPS) from Aeromonas salmonicida in Atlantic salmon, Salmo salar L.

The distribution of intravenously injected A-layer protein and lipopolysaccharide (LPS) purified from the outer surface of the fish pathogen Aeromonas salmonicida, was studied in Atlantic salmon. Radiolabelling was achieved by conjugating the antigens to tyramine cellobiose (TC) or fluorescein isothiocyanate (FITC) which were radioiodinated either before or after conjugation. Since both TC and FITC are trapped intralysosomally at the cellular site of uptake, the ligands are advantageous in studies on tissue distribution of antigens. Injection of TC-A-layer protein and TC-LPS resulted in high specific radioactivity (cpm g⁻¹tissue) in both head kidney and trunk kidney. In contrast, only low specific radioactivity was recovered in spleen, heart and liver. Surprisingly, use of FITC-LPS as the antigen changed the uptake to be high in both spleen and head kidney. Radiolabelled (¹²⁵I-TC-) LPS and A-protein, administered by a dorsal aorta catheterisation technique, were cleared from the blood within 24 h. In immunised fish, the antibody activity against the A-layer protein was diminished even within 10 min after administration, in contrast to the level of anti-LPS antibodies which remained high. These results suggest that immune-complex formation took place at least with the A-layer protein, but the uptake of A-layer protein in the various tissues did not differ significantly in vaccinated (A. salmonicida bacterin) and non-vaccinated fish.     

The efficiency of (Na + K)-ATPase in tumorigenic cells

The efficiency of (Na⁺ + K⁺)-ATPase (i.e. the amount of K⁺ pumped per ATP hydrolyzed) in intact tumorigenic cells was estimated in this study. This was accomplished by simultaneously measuring the rate of ouabain-sensitive K⁺ uptake and oxygen consumption in tumorigenic cell suspensions during the reintroduction of K⁺ to K⁺-depleted cells. The ATP turnover was then estimated by assuming 5.6–6 ATP/O₂ as the stoichiometry of NADH-linked respiration in these cells. In the three cell lines tested (hamster and chick embryo cells transformed with Rous sarcoma virus and Ehrlich ascites cells), the K⁺/ATP ratio was approximately 2, the same value as that found in normal tissues. Furthermore, only 20% of the total ATP production of these cells was used by (Na⁺ + K⁺)-ATPase.     

Molecular cloning of the plant plasma membrane H+-ATPase

Summary Mineral transport across the plasma membrane of plant cells is controlled by an electrochemical gradient of protons. This gradient is generated by an ATP-consuming enzyme in the membrane known as a proton pump, or H⁺-ATPase. The protein has a catalytic subunit of Mr=100,000 and is a prominent band when plasma membrane proteins are analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. We generated specific rabbit polyclonal antibody against the Mr=100,000 H⁺-ATPase and used the antibody to screen λgtll expression vector libraries of plant DNA. Several phage clones producing immunoreactive protein, and presumably containing DNA sequences for the ATPase structural gene, were isolated and purified from a carrot cDNA library and a Arabidopsis genomic DNA library. These studies represent our first efforts at cloning the structural gene for a plant plasma membrane transport protein. Applicability of the technique to other transport protein genes and the potential for use of recombinant DNA technology in plant mineral transport research are discussed.     

Utilization of nitrite and nitrate by dwarf bean

The uptake and conversion of NO ₂ ^- and the effect of NO ₂ ^- on the uptake and reduction of NO ₃ ^- were examined in N-depleted Phaseolus vulgaris L. Nitrite uptake at 0.1 mmol dm^-3 was against an electrochemical gradient and became constant after one or two initial phases. Steadystate uptake declined with increasing ambient NO ₂ ^- concentration (0–0.7 mmol dm^-3). In this concentration range root oxygen consumption was unaffected by NO ₂ ^- , indicating that the decrease of NO ₂ ^- uptake was not related to respiration. After 6 h NO ₂ ^- supply, about one-third of the absorbed NO ₂ ^- had accumulated, mainly in the root system. Oxidation of NO ₂ ^- to NO ₃ ^- was not observed. The apparent induction period for NO ₃ ^- uptake was about 6 h in control plants and 3.5 h in plants that were pretreated for 18 h with NO ₂ ^- . In contrast, the time course of NO ₂ ^- uptake was unaffected by pretreatment with NO ₃ ^- . Steadystate NO ₃ ^- uptake was less affected by NO ₂ ^- than was steady-state NO ₂ ^- uptake by NO ₃ ^- . Nitrate reductase activity (NRA) in leaves and roots was induced by both NO ₃ ^- and NO ₂ ^- . In roots, induction with NO ₂ ^- was faster than with NO ₃ ^- , but there was no difference in NRA after 5 h. Nitrite inhibited NRA in the roots of NO ₃ ^- -induced plants and thus seems to stimulate the induction, but not the activity of induced nitrate reductase. In view of the observed differences in time course and mutual competition, a common uptake mechanism for NO ₂ ^- and NO ₃ ^- seems unlikely. Expression of the NO ₂ ^- effect on the induction of NO ₃ ^- uptake required more time than the induction itself. We therefore conclude that NO ₂ ^- is not the physiological inducer of NO ₃ ^- uptake.Abbreviations NR(A) nitrate reductase (activity) - BM basal medium