Cytoplasmic free calcium in Riccia fluitans L. and Zea mays L.: Interaction of Ca2+ and pH?

In cells of Zea mays (root hairs, coleoptiles) and Riccia fluitans (rhizoids, thalli) intracellular Ca²⁺ and pH have been measured with double-barrelled microelectrodes. Free Ca²⁺ activities of 109–187 nM (Riccia rhizoids), 94–160 nM (Riccia thalli), 145–231 nM (Zea root hairs), 84–143 nM (Zea coleoptiles) were found, and therefore identified as cytoplasmic. In a few cases (Riccia rhizoids), free Ca²⁺ was in the lower millimolar range (2.3±0.8 mM). A change in external Ca²⁺ from 0.1 to 10 mM caused an initial and short transient increase in cytoplasmic free Ca²⁺ which finally levelled off at about 0.2 pCa unit below the control, whereas in the presence of cyanide the Ca²⁺ activity returned to the control level. It is suggested that this behaviour is indicative of active cellular Ca²⁺ regulation, and since it is energy-dependent, may involve a Ca²⁺-ATPase. Acidification of the cytoplasmic pH and alkalinization of the vacuolar pH lead to a simultaneous increase in cytoplasmic free Ca²⁺, while alkalinization of pH_c decreased the Ca²⁺ activity. Since this is true for such remote organisms as Riccia and Zea, it may be concluded that regulation of cytoplasmic pH and free Ca²⁺ are interrelated. It is further concluded that double-barrelled microelectrodes are useful tools for investigations of intracellular ion activities in plant cells.Symbols and abbreviations _m, _m membrane potential difference, changes thereof - PVC polyvinylchloride     

Cyclin-dependent kinase inhibitor,roscovitine, in combination with exogenous cytokinin,N6-benzyladenine,causes increase of cis-cytokinins in immobilized tobacco cells

Dynamics of the response of tobacco cells (line BY-2) to exogenous cytokinin, N ⁶-benzyladenine, and cyclin-dependent kinase inhibitor, roscovitine, was followed using alginate-immobilized cells packed into a column. N ⁶-Benzyladenine (1.25 M) increased the synthesis of the physiologically-active endogenous cytokinin, isopentenyladenosine, in the effluent up to 0.1 nM. Simultaneously, conversion of the excess of endogenous cytokinins to biologically inactive derivatives of cis-zeatin occurred, up to 0.8 nM. Roscovitine (50 M) further increased cis-cytokinins, up to 2.2 nM.     

Polyphenolic Antioxidants Efficiently Protect Urease from Inactivation by Ultrasonic Cavitation

Inactivation of urease (25 nM) in aqueous solutions (pH 5.0–6.0) treated with low-frequency ultrasound (LFUS; 27 kHz, 60 W/cm², 36–56°C) or high-frequency ultrasound (HFUS; 2.64 MHz, 1 W/cm², 36 or 56°C) has been characterized quantitatively, using first-order rate constants: k _in, total inactivation; k _in ^*, thermal inactivation; and k _in(us), ultrasonic inactivation. Within the range from 1 nM to 10 M, propyl gallate (PG) decreases by approximately threefold the rate of LFUS-induced inactivation of urease (56°C), whereas resorcinol poly-2-disulfide stops this process at 1 nM or higher concentrations. PG completely inhibits HFUS-induced inactivation of urease at 1 nM (36°C) or 10 nM (56°C). At 0.2–1.0 M, human serum albumin (HSA) increases the resistance of urease treated with HFUS to temperature- and cavitation-induced inactivation. Complexes of gallic acid polydisulfide (GAPDS) with HSA (GAPDS–HSA), formed by conjugation of 1.0 nM GAPDS with 0.33 nM HSA, prevent HFUS-induced urease inactivation (56°C).     

Synthetic peptide VKGFY and its cyclic analog stimulate macrophage bactericidal activity through non-opioid beta-endorphin receptors

We synthesized linear and cyclic pentapeptides corresponding to the sequence 369-373 of human immunoglobulin G heavy chain—VKGFY (referred to as pentarphin and cyclopentarphin, respectively). The effect of pentarphin and cyclopentarphin on phagocytosis of Salmonella typhimurium virulent 415 strain bacteria by mouse peritoneal macrophages in vitro was studied. Control experiments showed that macrophages actively captured these bacteria, but did not digest them: the captured microbes were viable and continued to proliferate inside the phagocytes; within 12 h all macrophage monolayer was destroyed (incomplete phagocytosis). If 1 nM pentarphin or cyclopentarphin was added to the cultivation medium, macrophage bactericidal activity was significantly increased and they digested all captured microorganisms within 6 h (complete phagocytosis). To study the receptor binding properties of pentarphin and cyclopentarphin we prepared ¹²⁵I-labeled pentarphin (179 Ci/mmol specific activity). The binding of ¹²⁵I-labeled pentarphin to mouse peritoneal macrophages was highaffinity (K _d = 3.6 ± 0.3 nM) and saturable. Studies on binding specificity revealed that this binding was insensitive to naloxone and [Met⁵]enkephalin, but completely inhibited by unlabeled cyclopentarphin (K _i = 2.6 ± 0.3 nM), immunorphin (K _i = 3.2 ± 0.3 nM), and -endorphin (K _i = 2.8 ± 0.2 nM). Thus, the effects of pentarphin and cyclopentarphin on macrophages are mediated by naloxone-insensitive receptors common for pentarphin, cyclopentarphin, immunorphin, and -endorphin.     

Growth,N2 fixation and photosynthesis in a cyanobacterium,Trichodesmium sp., under Fe stress

Trichodesmium sp., isolated from the Great Barrier Reef lagoon, was cultured in artificial seawater media containing a range of Fe concentration. Fe additions stimulated growth, N₂ fixation, cellular chlorophyll a content, light-saturated chlorophyll a-specific gross photosynthetic capacity (P_m ^chla) and the dark respiration rate (R_d ^chla). Cell yields only doubled for 9 nM Fe relative to zero added Fe, whereas N₂ fixation increased 11-fold considerably for 450 nM Fe. The results suggest that N₂ fixation of Trichodesmium is more sensitive to Fe limitation than are the cell yields.     

Relation between the anion exchange protein in kidney medullary collecting duct cells and red cell band 3

Summary A membrane protein that is immunochemically similar to the red cell anion exchange protein, band 3, has been identified on the basolateral face of the outer medullary collecting duct (MCD) cells in rabbit kidney. In freshly prepared separated rabbit MCD cells, M.L. Zeidel, P. Silva and J.L. Seifter (J. Clin. Invest. 77:1682–1688, 1986) found that Cl^–/HCO ₃ ^- exchange was inhibited by the stilbene anion exchange inhibitor, DIDS (4,4-diisothiocyano-2,2-disulfonic stilbene), with aK ₁ similar to that for the red cell. We have measured the binding affinities of a fluorescent stilbene inhibitor, DBDS (4,4-dibenzamido-2,2-disulfonic stilbene), to MCD cells in 28.5 mM citrate and have characterized both a high-affinity site (K ₁ ^s =93±24 mM) and a lower affinity site (K ₂ ^s =430±260 nM), which are closely similar to values for the red cell of 110±51 nM for the high-affinity site and 980±200 nM for the lower affinity site (A.S. Verkman, J.A. Dix & A.K. Solomon,J. Gen. Physiol. 81:421–449, 1983). When Cl^– replaces citrate in the buffer, the two sites collapse into a single one withK ₁ ^s =1500±400 nM, similar to the singleK ₁ ^s =1200±200 nM in the red cell (J.A. Dix, A.S. Verkman & A.K. Solomon,J. Membrane Biol. 89:211–223, 1986). The kinetics of DBDS binding to MCD cells at 0.25 M^–1 are characterized by a fast process, =0.14±0.03 sec, similar to =0.12±0.03 sec in the red cell. These similarities show that the physical chemical characteristics of stilbene inhibitor binding to MCD cell band 3 closely resemble those for red cell band 3, which suggests that the molecular structure is highly conserved.     

Kinetic analyses of waterborne Ca and Cd transport and their interactions in the gills of rainbow trout (<Emphasis Type="Italic">Oncorhynchus mykiss</Emphasis>) and yellow perch (<Emphasis Type="Italic">Perca flavescens</Emphasis>), two species differing greatly in acute waterborne Cd sensitivity

We evaluated the differential nature of interactions between waterborne Ca and Cd transport in the gills of yellow perch (Perca flavescens) and rainbow trout (Oncorhynchus mykiss), two species with a more than 400-fold difference in acute waterborne Cd tolerance. The J_max (maximum rate of uptake) and K_m (inverse of affinity) for Ca uptake, in the absence of Cd, were significantly lower in yellow perch (120.48 nM g^–1 wet wt h^–1 and 92.17 M, respectively) relative to rainbow trout (188.68 nM g^–1 wet wt h^–1 and 243.90 M, respectively). Similarly, the J_max for Cd uptake, at the lowest waterborne Ca level (100 M) tested, was significantly lower in yellow perch (0.27 nM g^–1 wet wt h^–1) relative to rainbow trout (0.40 nM g^–1 wet wt h^–1), but no significant difference was observed in the K_m values between the two species (yellow perch: 32.47 nM; rainbow trout: 31.27 nM). Waterborne Cd (0–890 nM) as well as waterborne Ca (100–1,000 M) competitively inhibited branchial uptake of each other in both species. However, analyses of inhibitor constants for branchial Ca uptake by waterborne Cd ( ) revealed that the inhibition was about 1.8 times more potent in rainbow trout compared to yellow perch. In contrast, analyses of inhibitor constants for branchial Cd uptake by waterborne Ca ( ) indicated that the inhibition was more than three fold more potent in yellow perch than in rainbow trout. Higher branchial Ca uptake and more potent inhibition by Cd as well as higher branchial Cd uptake and less potent inhibition by Ca were also reflected in whole-body measurements of Ca and Cd influx in trout relative to perch. Overall, whole-body effects were in accord with the branchial kinetic analyses. These results further strengthen the conclusion that branchial influxes of Ca and Cd occur through common pathways. Moreover, interspecific differences in acute waterborne Cd sensitivity can be explained, at least in part, by the differential nature of interactions between waterborne Ca and Cd transport in fish gills.Abbreviations FAAS flame atomic absorption spectrophotometer - GFAAS graphite furnace atomic absorption spectrophotometer - J _max maximum rate of uptake - K _i inhibitor constant - K _m substrate concentration at which the rate of uptake is half of the J_max - 96 h LC50 concentration at which 50% mortality occurs after 96 h Communicated by L.C.-H. WangThis revised version was published online in February 2004 with corrections to the abbreviation .     

Response of growth and photosynthesis of marine red tide alga Heterosigma akashiwo to iron and iron stress condition

Heterosigma akashiwo, a red tide alga, was grown in Fe-deficient and Fe-replete batch cultures. Cell final yields and the growth rate were limited when Fe was below 10 nM and alleviated with 100 nM Fe. By comparison with the results under Fe-replete conditions, chlorophyll a-specific and cell-specific light saturated net photosynthetic capacity (P_m ^{chl a} and P_m ^cell), dark respiration rate (R_d ^{chl a} and R_d ^cell) and apparent photosynthetic efficiency (^{chl a} and ^cell) decreased proportionately, whereas the cells became light saturated at higher irradiance under Fe stress (Fe-limited conditions).     

An [3H]oxotremorine binding method reveals regulatory changes by guanine nucleotides in cholinergic muscarinic receptors of cerebral cortex

A rapid, reliable filtration method for [³H]oxotremorine binding to membranes of the cerebral cortex that allows the direct study of regulation by guanine nucleotides of muscarinic receptors was developed. [³H]Oxotremorine binds to cerebral cortex membranes with high affinity (K _D, 1.9 nM) and low capacity (B _max, 187 pmol/g protein). These sites, which represent only about 18% of those labeled with [³H]quinuclidinyl benzilate, constitute a population of GTP-sensitive binding sites. Association and dissociation binding experiments revealed a similar value ofK _D (2.3 nM). Displacement studies with 1–4000 nM oxotremorine showed the existence of a second binding site of low affinity (K _D, 1.2 M) and large capacity (B _max, 1904 pmol/g protein). Gpp(NH)p, added in vitro, produced a striking inhibition of [³H]oxotremorine binding with an IC 50 of 0.3 M. Saturation assays, in the presence of 0.5 M Gpp(NH)p, revealed a non-competitive inhibition of the binding with little change in affinity. These results are discussed from the viewpoint of conflicting reports in the literature about guanine nucleotide regulation of muscarinic receptors in reconstituted systems and membranes from different tissues.     

Honokiol and Magnolol Increase the Number of [3H]Muscimol Binding Sites Three-Fold in Rat Forebrain Membranes In Vitro Using a Filtration Assay,by Allosterically Increasing the Affinities of Low-Affinity Sites

1. The bark of the root and stem of various Magnolia species has been used in Traditional Chinese Medicine to treat a variety of disorders including anxiety and nervous disturbances. The biphenolic compounds honokiol (H) and magnolol (M), the main components of the Chinese medicinal plant Magnolia officinalis, interact with GABA_A receptors in rat brain in vitro. We compared the effects of H and M on [³H]muscimol (MUS) and [³H]flunitrazepam (FNM) binding using EDTA/water dialyzed rat brain membranes in a buffer containing 150 mM NaCl plus 5 mM Tris-HCl, pH 7.5 as well as [³⁵S]t-butylbicyclophosphorothionate (TBPS) in 200 mM KBr plus 5 mM Tris-HCl, pH 7.5. H and M had similar enhancing effects on [³H]MUS as well as on [³H]FNM binding to rat brain membrane preparations, but H was 2.5 to 5.2 times more potent than M. 2. [ ³ H]FNM binding. GABA alone almost doubled [³H]FNM binding with EC₅₀ = 450 nM and 200 nM using forebrain and cerebellar membranes, respectively. In the presence of 5 M H or M the EC₅₀ values for GABA were decreased to 79 and 89 nM, respectively, using forebrain, and 39 and 78 nM, using cerebellar membranes. H and M potently enhanced the potentiating effect of 200 nM GABA on [³H]FNM binding with EC₅₀ values of 0.61 M and 1.6 M using forebrain membranes, with maximal enhancements of 33 and 47%, respectively. Using cerebellar membranes, the corresponding values were 0.25 and 1.1 M, and 22 and 34%. 3. [ ³ H]MUS binding. H and M increased [³H]MUS binding to whole forebrain membranes about 3-fold with EC₅₀ values of 6.0 and 15 M. Using cerebellar membranes, H and M increased [³H]MUS binding ~68% with EC₅₀ values of 2.3 and 12 M, respectively. Scatchard analysis revealed that the enhancements of [³H]MUS binding were due primarily to increases in the number of binding sites (B_max values) with no effect on the high affinity binding constants (K_d values). The enhancing effect of H and M were not additive. 4. [ ³⁵ S]TBPS binding. H and M displaced [³⁵S]TBPS binding from sites on whole rat forebrain membranes with IC₅₀ values of 7.8 and 6.0 M, respectively. Using cerebellar membranes, the corresponding IC₅₀ values were 5.3 and 4.8 M. These inhibitory effects were reversed by the potent GABA_A receptor blocker R5135 (10 nM), suggesting that H and M allosterically increase the affinity of GABA_A receptors for GABA and MUS by binding to sites in GABA_A receptor complexes. 5. Two monophenols, the anesthetic propofol (2,6-diisopropylphenol, P) and the anti-inflammatory diflunisal (2,4-difluoro-4-hydroxy-3-biphenyl carboxylic acid, D) also enhanced [³H]MUS binding, decreased the EC₅₀ values for GABA in enhancing [³H]FNM binding and potentiated the enhancing effect of 200 nM GABA on [³H]FNM binding, although enhancements of [³H]MUS binding for these monophenols were smaller than those for H and M, using forebrain and cerebellar membranes. The enhancing effect of P and D on [³H]MUS binding were almost completely additive. 2,2-biphenol was inactive on [³H]MUS and [³H]FNM binding. These, and other preliminary experiments, suggest that appropriate ortho (C2) and para (C4) substitution increases the GABA-potentiating activity of phenols. 6. The potentiation of GABAergic neurotransmission by H and M is probably involved in their previously reported anxiolytic and central depressant effects.     

A high-affinity folate binding protein in human amniotic fluid. Radioligand binding characteristics,immunological properties and molecular size

The presence of a folate binding protein of high-affinity type (affinity constant 5 · 10⁹M^–1, maximum folate binding 3 nM) in human amniotic fluid was demonstrated in equilibrium dialysis experiments (37°C, pH 7.4) with the radioligand³H-folate. Dissociation of³H-folate from the binding protein was slow at pH 7.4 but rapid at pH 3.5. By use of rabbit antibodies against low molecular weight folate binding protein from human milk we determined the concentration of folate binding protein in 5 amniotic fluids (range 1.5–2.3 nM) in an Enzyme-Linked Immunosorbent Assay (ELISA). ultrogel AcA 44 chromatography of amniotic fluid showed that immunoreactive and radioligand bound folate binding protein coeluted in two peaks: a major one (M _r 25 000) and a minor one (M _r 100 000).     

Effect of arterial perfusion on net water flux and active sodium transport across the isolated skin ofBufo bufo bufo (L.)

Summary A method has been developed which allows perfusion of the blood vessels in isolated pelvic skins ofB. bufo. The effect of various doses of vasotocin (AVT) on net water flux (inside medium 220 mOsM, outside medium 11 mOsM) and active sodium transport were compared in perfused and unperfused skins. The unstimulated water flux (Fig. 3) and the active sodium transport (Fig. 6) were unaffected by perfusion. The threshold for stimulation of water flux was between 0.01 and 0.1 nM vasotocin in perfused skins and between 0.1 and 1 nM in unperfused skins. The threshold for stimulation of active sodium transport was between 0.005 and 0.05 nM vasotocin in perfused skins and between 0.05 and 0.5 nM in unperfused skins. The maximal water flux through perfused skins, 4 l/cm²min, was obtained at 1 nM vasotocin. At 10 nM vasotocin the water flux was only 0.7 l/cm²min in unperfused skins. The maximal active sodium transport was approximately of the same magnitude in perfused and unperfused skins, at 0.5 mM and at 50 nM, respectively.     

Cation Transport Specificity at the Blood–Brain Barrier

The molecular identification, expression and cloning of membrane-bound organic cation transporters are being completed in isolated in vitro membranes. In vivo studies, where cation specificity overlaps, need to complement this work. Method: Cross-inhibition of [³H]choline and [³H]thiamine brain uptake by in situ rat brain perfusion. Results: [³H]Choline brain uptake was not inhibited by thiamine at physiologic concentrations (100 nM). However, choline ranging from 100 nM to 250 M inhibited [³H]thiamine brain uptake, though not below levels observed at thiamine concentrations of 100 nM. Conclusion: (1) The molecular family of the blood–brain barrier (BBB) choline transporter may be elucidated in vitro by its interaction with physiologic thiamine levels, and (2) two cationic transporters at the BBB may be responsible for thiamine brain uptake.     

Effect of Synthetic β-Endorphin-Like Peptide Immunorphin on Human T Lymphocytes

-Endorphin and the synthetic -endorphin-like decapeptide Ser-Leu-Thr-Cys-Leu-Val-Lys-Gly-Phe-Tyr (referred to as immunorphin), corresponding to the sequence 364-373 of the CH3 domain of human immunoglobulin G heavy chain, were shown to stimulate concanavalin A-induced proliferation of T lymphocytes from the blood of healthy donors. [Met⁵]Enkephalin and the antagonist of opioid receptors naloxone examined in parallel were inactive. The stimulating effect of -endorphin and immunorphin on T lymphocyte proliferation is not inhibited by naloxone. Studies on receptor binding of ¹²⁵I-labeled immunorphin to T lymphocytes revealed that it binds with high affinity to naloxone-insensitive receptors (K _d = 7.0 ± 0.3 nM)). Unlabeled immunorphin completely inhibits ¹²⁵I-labeled -endorphin specific binding to naloxone insensitive receptors on T lymphocytes (K _i = 0.6 ± 0.1 nM)). Thus, -endorphin and immunorphin interact with common naloxone insensitive receptors on T lymphocytes.     

GABA-agonists induce the formation of low-affinity GABA-receptors on cultured cerebellar granule cells via preexisting high affinity GABA receptors

The kinetics of specific GABA-binding to membranes isolated from cerebellar granule cells, cultured for 12 days from dissociated cerebella of 7-day-old rats was studied using [³H]GABA as the ligand. The granule cells were cultured in the presence of the specific GABA receptor agonist 4, 5, 6, 7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP, 150 M) or THIP plus the antagonist bicuculline methobromide (150 M of each) or in the absence of the agonist or antagonist. Membranes isolated from granule cells cultured in a medium without the GABA agonist revealed a single binding site for GABA with a binding constant (K _D) of 7.9±0.4 nM and aB _max of 3.42±0.08 pmol×mg^–1 protein. Membranes from cells cultured in the presence of THIP had two binding sites for GABA withK _D-values of 6.8±0.9 nM and 476±311 nM, respectively. The correspondingB _max values were 4.41±0.42 pmol×mg^–1 and 5.81±1.20 pmol×mg^–1. The effect of culturing the cells in THIP was antagonized by the simultaneous presence of bicuculline in the culture media, i.e. no significant low-affinity binding for GABA was found on the membranes from granule cells cultured in both THIP and bicuculline. TheK _D value (14.3±1.4 nM) for the high affinity binding site was, however, slightly increased compared to the non-treated cells. These findings suggest that the ability of THIP to induce formation of low-affinity GABA receptors is mediated by preexisting high-affinity GABA-receptors on the granule cells.     

Phorbol ester and the actions of phosphatidylinositol 4,5-bisphosphate specific phospholipase C and protein kinase C in microsomes prepared from cultured cardiomyocytes

Microsomes were prepared from cultured neonatal rat cardiomyocytes. Incubation of microsomes in buffer containing 5µM CaCl₂, 5 mM cholate and 100 nM [3H-]Phosphatidylinosito14,5-bisphosphate (PtdIns(4,5) P₂) resulted in the formation of [³H-]InsP ₃. GTP-gamma-S (125 µM) stimulated the production of [³H-]InsP ₃. Microsomes prepared from phorbol ester-treated (100 nM phorbol 12-myristate 13-acetate, PMA) cardiomyocytes showed decreased activities of basal as well as GTP-gamma-S-stimulated [³H-]Ptdlns(4,5)P ₂ hydrolysis. In the microsomes a 15 kD protein was demonstrated to be the major substrate phosphorylated by intrinsic protein kinase C, which was activated by 0.5 mM Ca²⁺. Addition of phorbol ester (100 nM PMA) enhanced the ³²P-incorporation into the 15 kD protein. Protein kinase C, purified from rat brain, in the presence of Ca²⁺, diglyceride, and phosphatidylserine did not change the phosphorylation pattern any further. In conclusion, it was shown that phorbol ester pretreatment of neonatal rat cardiomyocytes reduces microsomel GTP-gamma-S-stimulated Ptdlns(4,5)P ₂-specific phospholipase C activity, as estimated with exogenous substrate, and that in cardiomyocyte microsomes phorbol ester activates protein kinase C-induced 15 kD protein phosphorylation. The results indicate that phorbol ester may down-regulate -adrenoceptor mediated Ptdlns(4,5)P ₂ hydrolysis by activation of protein kinase C-induced 15 kD protein phosphorylation.List of abbreviations ATP Adenosine 5-Trphosphate - CSU Catalytic Subunit of cyclic AMP-dependent protein kinase - DG Diacylglycerol - DMSO Dimethylsulfoxide - DTT DL-dithiothreitol - EDTA Ethylenedinitrilotetraacetic Acid - EGTA Ethyleneglycol-0,0-bis(aminoethyl)-N,N,N,N,-tetraacetic acid - GTP-gamma-S Guanosine 5-O-(3-thiotriphosphate) - HPTLC High Performance Thin Layer Chromatography - InsP ₃ Inositol monophosphate - InsP ₂ Inositol bisphosphate - InsP ₃ Inositol trisphosphate - MES 2-Morpholinoethanesulfonic acid - MOPS 3-[N-Morpholino]Propanesulfonic acid - PAGE Polyacrylamide-gel Electrophoresis - PKC Protein Kinase C - PLase C Phospholipase C - PMA Phorbol 12-Myristate 13-Acetate - PMSF Phenylmethylsulfonyl Fluoride - PtdSer Phosphatidylserine - PtdIns Phosphatidyl inositol - PT Pertussis Toxin - Ptdlns(4)P Phosphatidylinositol 4-monophosphate - Ptdlns (4,5)PZ-Phosphatidylinositol4,5-bisphosphate - SDS-Sodium Dodecyl Sulfate Tris-Tris(hydroxymethyl) aminomethane     

Inactivation of a newly transferred gene in recombination-deficient recipients of Escherichia coli

Summary The expression of a newly transferred lacZ ⁺ gene in lacZ recipients carrying various mutations in the recA and recB genes was studied by measuring the rates of induced synthesis of -galactosidase in zygotes formed after mating with either F or Hfr donors. The ability to synthesize -galactosidase decreases with time in both recA and recB zygotes when the lacZ ⁺ gene is transferred from an Hfr donor, but not when the lacZ gene is transferred from an F donor. There is no such inactivation of the newly transferred lacZ ⁺ gene in Rec⁺ zygotes. We conclude that the functioning of the transferred DNA is progressively inactivated in rec recipients unless the DNA is contained in an episome such as F.     

Contribution of de novo protein synthesis to the hypertrophic effect of IGF-1 but not of thyroid hormones in adult ventricular cardiomyocytes

Background: Enhanced expression of IGF-1 occurs in left ventricular hypertrophy (LVH) associated with systemic hypertension. Cardiac dysfunction accompanied by LVH is also observed in hyperthyroidism. Objective: to assess the relative contributions of de novo protein synthesis and attenuated protein degradation to increased protein mass associated with cardiomyocyte hypertrophy elicited by IGF-1 and thyroid hormones (tri-iodo thyronine T₃, and l-thyroxine T₄), respectively. Methods: total mass of protein, and both the incorporation, and removal of previously incorporated l-U-¹⁴C-phenylalanine, indices of protein synthesis and degradation, respectively, were assessed in quiescent adult rat ventricular cardiomyocytes maintained in short-term culture, and corrected for DNA content, as a index of cell number. Results: IGF-1 (1 pM-100 nM) increased cell protein significantly, maximally at 1 nM and by 38% above basal value after 24 h. T₃ (10 pM-2 M) and T₄ (10 pM-2 M) increased cell protein significantly maximally at 1 M and by 33.2 and 30.5%, respectively, above basal value. IGF-1 ( 10 pM), T₃ (10 pM-2 M) and T₄ (10 pM-2 M) did not increase incorporation of l-U-₁₄C-phenylalanine above basal values. IGF-1 (100 pM-100 nM) increased incorporation of radiolabel significantly maximally at 100 nM and by 56%. T₄ (100 pM) and IGF-1 (10 pM), concentrations that did not stimulate de novo protein synthesis, attenuated the degradation of radiolabelled protein by 13.6 and 11.8%, respectively, compared to control values after 48 h. Conclusion: These data indicate that the acute hypertrophic response to (i) thyroid hormones cannot be attributed to initiation of de novo protein synthesis; (ii) IGF-1 comprises two components; the response elicited by IGF-1 (< 10 pM) is independent of, while the response elicited by IGF-1 (> 100 pM) is due to de novo protein synthesis.     

G protein dependent alterations in [125I]iodocyanopindolol and±cyanopindolol binding at 5-HT1B binding sites in rat brain membranes

Several manipulations that affect G protein/receptor coupling also alter the binding of [¹²⁵I]iodocyanopindolol ([¹²⁵I]ICYP)±cyanopindolol (±CYP) to rat brain 5-HT_1B binding sites in radiologand binding assays. Inclusion of 5 mM MgSO₄ in these assays results in a small but significant increase in the affinity of [¹²⁵I]ICYP (fromK _D=0.046 nM toK _D=0.037 nM). In contrast, 100 M Gpp(NH)p, GTP, or GDP reduce [¹²⁵I]ICYP affinity (K _D=0.056 nM with GTP) while ATP and GMP are less effective.±CYP affinity for 5-HT_1B sites labeled by [³H]dihydroergotamine ([³H]DE) also displays a small but significant reduction (from K_i=1.4 nM to K_i=3.5nM) by the inclusion of 100 M GTP. Pre-treatment of the brain membranes with N-ethylmaleimide (NEM) in concentrations known to inactivate many G proteins reduces 5-HT_1B specific binding of [¹²⁵I]ICYP. The NEM induced reduction in [¹²⁵I]ICYP binding can be reversed by reconstitution with purified exogenous G proteins (Go and Gi), demonstrating directly that high affinity binding of [¹²⁵I]ICYP to 5-HT_1B sites is dependent on G proteins. The effects of Mg²⁺ ion, guanine nucleotides, NEM and G protein reconstitution on [¹²⁵I]ICYP and ±CYP binding are all hallmarks of agonist binding to G protein linked receptors. The effect of GTP, however, is quantitatively much less for the binding of these pindolol derivatives than for the binding of 5-HT, a presumed full agonist at 5-HT_1B sites. The relatively slight stabilization of [¹²⁵I]ICYP and ±CYP binding conferred by G protein/5-HT_1B receptor interaction may reflect the molecular events underlying previous observations that these compounds are partial 5-HT_1B agoinists.     

Properties of the thymidine transport system of chinese hamster ovary cells as probed by nitrobenzylthioinosine

Summary The transport of thymidine into Chinese hamster ovary cells grown in suspension culture was measured under conditions in which thymidine was not metabolized, namely, when cells had been depleted of ATP. The system transporting thymidine was saturable (K _m ^zt =70M), rapid (50% of transmembrane equilibrium level attained within 8 sec), and was apparently shared by other nucleosides, but not thymine or hypoxanthine. 6([4-nitrobenzyl]thio)-9--d-ribofuranosylpurine, nitrobenzylthioinosine, inhibited thymidine transport in a simple, noncompetitive fashion with an apparentK _i =1.0 nM (based on total concentration of inhibitor, which significantly overestimates that of free inhibitor). The rate of expression of inhibition was slow (t _1/2=17 sec) relative to the rate of association of thymidine with its transporter, and thymidine partially protected the transport system against inhibition by nitrobenzylthioinosine. The dissociation constant for the inhibitortransporter complex was estimated at about 0.1 nM, and the number of binding sites per cell at about 6×10⁴. HeLa, P388 murine leukemia, and mouse L cells were as sensitive to nitrobenzylthioinosine inhibition of thymidine transport as Chinese hamster ovary cells; Novikoff rat hepatoma cells were much less sensitive.