Chromatography of human urinary erythropoietin and granulocyte colony-stimulating factor on insolubilized phytohaemagglutinin.

Human urinary erythropoetin was absorbed to phytohaemagglutinin coupled to agarose or porous glass and quatitatively eluted by a saturated solution of MgCl2. This method provides a means of separating erythropoietin from several of its contaminants, presumably on the basis of its carbohydrate side chains. Erythropoietin which had been purified by chromatography on insolubilized phytohaemagglutinin was sufficiently free of toxicity to be assayable in tissue culture even when crude urine was used as a starting material.     

Hypoxia enhances prostaglandin synthesis in renal mesangial cell cultures

In view of recent findings which suggest that renal prostaglandins mediate the effect of hypoxia on erythropoietin production, we have studied whether hypoxia is a stimulus for in vitro prostaglandin synthesis. Studies were carried out in rat renal mesangial cell cultures which produce erythropoietin in an oxygen-dependent manner. Production rates of PGE₂ and in specified samples also of 6-keto-PGF_1α, as a measure of PGI₂, and PGF_2α were determined by radioimmunoassay after incubation at either 20% O₂ (normoxic) or 2% O₂ (hypoxic) in gas permeable dishes for 24 hrs. Considerable variation in PGE₂ production was noted among independent cell lines. PGE₂ production appeared to be inversely correlated to the cellular density of the cultures. In addition, PGE₂ production was enhanced in hypoxic cell cultures. The mean increase was 50 to 60%. PGF_2α and 6-keto-PGF_1α increased by about the same rate. These results indicate that hypoxia is a stimulus for in vitro prostaglandin production.     

The ionic control of 1,25-dihydroxyvitamin D-3 synthesis in isolated chick renal mitochondria: The role of potassium

In previous studies it was found that change in the concentrations of Ca²⁺, H⁺, and HPO₄²⁻ in the incubation medium altered the rates of synthesis of 1,25-dihydroxyvitamin D-3 (1,25(OH)₂D-3) by isolated renal mitochondria obtained from D-deficient chicks. The present studies demonstrate that raising the medium concentration of K⁺ from 1 to 50 mM leads to a 6-fold increase in rate of 1,25(OH)₂D-3 synthesis by isolated chick mitochondria; that the magnitude of this K⁺-dependent stimulation is enhanced by optimal concentrations of calcium (pCa = 5) and phosphate (pP_i = 3) (3 mM) but not by pH (from 6.8 to 7.4); that the effect is not produced by similar changes in media Na⁺ concentration; and that the stimulatory effect of K⁺ is not blocked by ruthenium red, an inhibitor of calcium transport and of the calcium-dependent stimulation of mitochondrial 1,25(OH)₂D-3 synthesis. It was also found taht valinomycin, a K⁺-specific ionophore, enhanced the sensitivity of the mitochondrial 1α-hydroxylase activity to K⁺. In the presence of valinomycin, an increase of pK⁺ to 3 was sufficient to cause a significant stimulation of 1,25(OH)₂D-3 synthesis. It was concluded that changes in the ion content of the mitochondrial matrix space regulate the activity of the 1α-hydroxylase.     

Taenia crassiceps in the rat: differences in susceptibility to infection and development of immunocompetence in relation to age and host strain.

The influence of host strain and age upon susceptibility of rats to infection with Taenia crassiceps metacestodes was examined. The polyclonal T-cell activators concanavalin-A and phytohaemagglutinin were used to demonstrate that the onset of host resistance to infection with the cestode is coincident with the acquisition of responsiveness to mitogens in the post-natal maturation of the AS₂ rat.     

Selenocysteine lyase activity in a cysteine-requiring mutant ofEscherichia coli K-12

Selenocysteine lyase activity was detected in crude extracts from a cysteine-requiring mutant ofEscherichia coli K-12. The level of activity was the same whether cells had been grown aerobically or anaerobically, with or without selenocysteine. Selenocysteine lyase catalyzes the conversion of selenocysteine to alanine and elemental Se, a reaction that is followed by a nonenzymatic reduction of the Se to hydrogen selenide. Both of these end products were identified in this study. With cysteine as the substrate, alanine and H₂S were formed, but only at levels 50% less than the products formed from selenocysteine. Selenocysteine lyase has been identified in a number of mammals and bacteria; its presence in a cysK mutant ofE. coli K-12 suggests a common route whereby hydrogen selenide, derived from selenocysteine, can then be assimilated into selenoproteins.     

Changes in the carbohydrate metabolism of mitogenically stimulated human peripheral lymphocytes : II. Relative importance of glycolysis and oxidative phosphorylation on phytohaemagglutinin stimulation

The effect of inhibition of the oxidative phosphorylation of human blood lymphocytes in the presence and absence of phytohaemagglutinin has been investigated. It was found that the incorporation of inorganic phosphate into acid-soluble nucleotides is dependent on, though not a direct measure of oxidative phosphorylation. Optimal concentration for inhibition of oxidative phosphorylation with oligomycin and the uncoupler 4,5,6,7-tetrachloro-2-trifluoromethylbenzimidazole (TTFB) were determined. Under conditions of maximally inhibited P_i incorporation into acid-soluble nucleotides (80%) and maximally increased oxygen consumption and lactate production (4–5 times), the stimulatory effect of phytohaemagglutinin on several glycolytic parameters could still be observed. Therefore, stimulation of cellular processes by PHA is still possible when energy is provided by glycolysis only.     

Active human erythropoietin expressed in insect cells using a baculovirus vector: A role for N-linked oligosaccharide

Biologically active recombinant human erythropoietin has been expressed at high levels in an insect cell background. Expression involved the preparation of a human erythropoietin cDNA, the transfer of this cDNA to the Autographa californica nuclear polyhedrosis virus (AcNPV) genome under the polyhedrin gene promoter, and the subsequent infection of Spodoptera frugiperda cells with recombinant AcNPV. Erythropoietin cDNA was prepared through the expression of the human erythropoietin gene in COS cells using pSV2 and the construction of a COS cell cDNA library in bacteriophage Lambda GT10. Prior to transfer to the AcNPV genome, erythropoietin cDNA isolated from this library was modified at the 3′-terminus in order to replace genomic erythropoietin for SV40 cDNA derived from pSV2. Transfer of this cDNA to AcNPV and the infection of S. frugiperda cells with cloned recombinant virus led to the secretion of erythropoietin: based on bioassay, rates of hormone secretion (over 40 U/ml per h) were 50-fold greater than observed for COS cells. The purified recombinant product possessed full biological activity (at least 200000 U/mg), but was of lower M_r (23000) than human erythropoietin produced in COS cells (30000) or purified from urine (30000 to 38000). This difference was attributed to the glycosylation of erythropoietin in S. frugiperda cells with oligosaccharides of only limited size. Further removal of N-linked oligosac-charides from this M_r 23000 hormone using N-Glycanase yielded an apo-erythropoietin (M_r 18000) which possessed substantially reduced biological activity. These results indicate that glycosylation, but not the normal processing of oligosaccharides to complex types, is required for the full hormonal activity of human erythropoietin during red cell development.     

Half channels mediating H transport and the mechanism of gating in the Fo sector of Escherichia coli F1Fo ATP synthase

H⁺-transporting F₁F_o ATP synthase catalyzes the synthesis of ATP via coupled rotary motors within F_o and F₁. H⁺ transport at the subunit a–c interface in trans-membranous F_o drives rotation of the c-ring within the membrane, with subunit c being bound in a complex with the γ and ε subunits extending from the membrane. Finally, the rotation of subunit γ within the α₃β₃ sector of F₁ mechanically drives ATP synthesis within the catalytic sites. In this review, we propose and provide evidence supporting the route of proton transfer via half channels from one side of the membrane to the other, and the mechanism of gating H⁺ binding to and release from Asp61 of subunit c, via conformational movements of Arg210 in subunit a. We propose that protons are gated from the inside of a four-helix bundle at the periplasmic side of subunit a to drive protonation of cAsp61, and that this gating movement is facilitated by the swiveling of trans-membrane helices (TMHs) 4 and 5 at the site of interaction with cAsp61 on the periphery of the c-ring. Proton release to the cytoplasmic half channel is facilitated by the movement of aArg210 as a consequence of this proposed helical swiveling. Finally, release from the cytoplasmic half channel is mediated by residues in a complex of interacting extra-membraneous loops formed between TMHs of both subunits a and c. This article is part of a Special Issue entitled: 18th European Bioenergetic Conference.     

GABA and glutamate fluxes in developing nerve endings

Radiolabeled GABA and glutamate transport into 7 day, 14 day and adult cortical nerve ending preparations was examined. Transport was measured at several Na⁺ concentrations, 19, 27, 43 and 121 mM, and at two temperatures, 15 and 30°C. K_m and V_max values were calculated for all experimental conditions by means of Wilkinson (1961) analysis. A comparison of the day 14 and adult data shows higher K_m values at all Na⁺ concentrations on day 14 for both GABA and glutamate transport. In addition, the temperature dependence of transport was attenuated in the day 14 preparation. Finally, the specificity of GABA transport, as measured by the use of the transport inhibitors β-alanine and 2,4-diaminobutyric acid, was not different between the day 14 and adult preparations. Overall, it is concluded that both GABA and glutamate transport into day 14 nerve endings behave as if “adult” transporter molecules were existing in a more fluid lipid environment, which is the situation found in synaptic membranes prepared from day 14 nerve endings (Hitzemann and Johnson, 1983).Glutamate and GABA transport into 7 day nerve endings is complex and shows marked differences from the day 14 and adult data. Day 7 GABA transport was significantly more sensitive to β-alanine inhibition. Day 7 transport was more sensitive to Na⁺ manipulation and the temperature dependent kinetics show unique Na⁺ effects not seen in the day 14 or adult preparations. For example, at 19 mM Na⁺, 7 day glutamate transport was more temperature dependent than adult transport but as the Na⁺ concentration was increased the reverse was true. The opposite situation for temperature-Na⁺ effects was seen for GABA transport. Finally, no Ca⁺²-dependent component of GABA release could be found in 7 day nerve endings while a significant component was found at day 14. Overall, it is concluded that both glutamate and GABA fluxes in 7 day nerve endings differ both qualitatively from that seen in both day 14 and adult nerve endings.     

STUDY OF THE EFFECT OF HYDROXYUREA ON THE ERYTHROPOIETIN-SENSITIVE CELLS

The response of polycythaemic mice to a standard dose of erythropoietin has been measured at various, time intervals after single or repeated injections of hydroxyurea. The results exclude S phase of the cell cycle as the period responsive to erythropoietin. They suggest the existence of feedback mechanisms within the cell cycle, operating at the G₁-S boundary and within the G₁ phase. Hydroxyurea given to polycythaemic mice at various time intervals after erythropoietin induced characteristic changes in the response. These changes can be explained if both gradual transit of differentiated cells into the DNA synthesis (S phase) and changes in amount of the erythropoietin sensitive cells caused by the feedback mechanisms operating in the cell cycle are considered.     

Enhanced survival of lethally irradiated adenosine A3 receptor knockout mice. A role for hematopoietic growth factors?

Adenosine A₃ receptor knockout (A₃AR KO) mice and their wild-type (WT) counterparts were compared from the point of view of their abilities to survive exposures to lethal doses of γ-radiation belonging to the range of radiation doses inducing the bone marrow acute radiation syndrome. Parameters of cumulative 30-day survival (experiment using a midlethal radiation dose) or cumulative 11-day survival (experiment using an absolutely lethal radiation dose), and of mean survival time were evaluated. The values of A₃AR KO mice always reflected their higher survival in comparison with WT ones, the P values being above the limit for statistical significance after the midlethal radiation dose and standing for statistical significance after the absolutely lethal radiation dose. This finding was considered surprising, taking into account the previously obtained findings on defects in numbers and functional properties of peripheral blood cells in A₃AR KO mice. Therefore, previous hematological analyses of A₃AR KO mice were supplemented in the present studies with determination of serum levels of the granulocyte colony-stimulating factor, erythropoietin, and thrombopoietin. Though distinct differences in these parameters were observed between A₃AR KO and WT mice, none of them could explain the relatively high postirradiation survival of A₃AR KO mice. Further studies on these mice comprising also those on other than hemopoietic tissues and organs can help to clarify their relative radioresistance.     

Dietary Protein-induced Changes of the Erythropoietin Level in Rat Serum

Erythropoietin, a glycoprotein, is the primary regulator of erythropoiesis. The most convenient and sensitive assay for active erythropoietin is to measure its stimulatory effect on in vitro ³H-thymidine incorporation into DNA of erythropoietin-responsive cells. An attempt with this method to estimate the erythropoietin level in rat serum, however, was unsuccesful because of the presence of inhibitory substance(s) and non-erythropoietic factor(s) stimulating ³H-thymidine incorporation. Pretreatment of the serum by heating, extraction of erythropoietin from denatured-protein aggregates, and subsequent concentration of erythropoietin in the extract with alcohol precipitation made it possible to measure the serum erythropoietin levels. Rabbit anti-erythropoietin antibody was used for a quantitative estimation of erythropoietin in the concentrated extracts. Erythropoietin levels in sera of rats fed on varied amounts of casein for 7 days were measured with these procedures to find if the impairment of erythropoiesis upon protein deprivation was due to changes in the erythropoietin level. We found that the level in protein-deprived rats was less than 1/8 that of 20% casein-fed rats, a level undetectable by the present assay, and that the serum erythropoietin increased as the protein content in the diet was increased up to 20%, then leveled off. The erythropoietin in serum decreased rapidly after protein deprivation; the level at 12hr after deprivation began was about 1/5 that in 20% casein-fed rats. Thus, the depression of erythropoiesis upon protein deprivation is primarily caused by the lowered level of erythropoietin.     

Expression and analysis of the glycosylation properties of recombinant human erythropoietin expressed in Pichia pastoris

The Pichia pastoris expression system was used to produce recombinant human erythropoietin, a protein synthesized by the adult kidney and responsible for the regulation of red blood cell production. The entire recombinant human erythropoietin (rhEPO) gene was constructed using the Splicing by Overlap Extension by PCR (SOE-PCR) technique, cloned and expressed through the secretory pathway of the Pichia expression system. Recombinant erythropoietin was successfully expressed in P. pastoris. The estimated molecular mass of the expressed protein ranged from 32 kDa to 75 kDa, with the variation in size being attributed to the presence of rhEPO glycosylation analogs. A crude functional analysis of the soluble proteins showed that all of the forms were active in vivo.     

Studies of the incorporation of [5-3H]uridine during activation and transformation of lymphocytes induced by phytohaemagglutinin. Dependence of the incorporation rate on uridine concentration at certain critical concentrations

1. Partially purified pig blood lymphocytes were stimulated to transform in culture with phytohaemagglutinin. Initial cell activation was assessed by measuring the increase of uridine incorporation into RNA induced by phytohaemagglutinin. The phytohaemagglutinin/serum ratio for this effect was similar to that required for transformation; however, no inhibition at high phytohaemagglutinin/serum ratios was found during cell activation. 2. Without replenishment of medium the pool of competitors with added uridine for incorporation fell to zero during 2 days of culture. At certain critical pool concentrations uridine itself could stimulate the rate of uridine incorporation. 3. Most of the tritium from [5-(3)H]uridine added at the initiation of culture had been incorporated into RNA by the end of the second day of culture; the subsequent loss of radioactivity preceded a fall in the total RNA content of cultures. 4. RNA was qualitatively examined on sucrose density gradients. In the first 30min. after the addition of phytohaemagglutinin, increased labelling occurred predominantly between the 28s and 4s regions of the gradients. On the second day of culture with phytohaemagglutinin mainly RNA sedimenting beyond the 28s region of gradients was labelled in 30min.     

Imidazo[1,2-α]pyridines possess adenosine A1 receptor affinity for the potential treatment of cognition in neurological disorders

Previous research has shown that bicyclic 6:5-fused heteroaromatic compounds with two N-atoms have variable degrees of adenosine A₁ receptor antagonistic activity. Prompted by this imidazo[1,2-α]pyridine analogues were synthesized and evaluated for their adenosine A₁ and A_2A receptor affinity via radioligand binding studies and subjected to a GTP shift assay to determine its adenosine A₁ receptor agonistic or antagonistic functionality. Imidazo[1,2-α]pyridine, the parent scaffold, was found devoid of affinity for the adenosine A₁ and A_2A receptors. The influence of substitution on position C2 showed no improvement for either adenosine A₁ or A_2A receptor affinity. The addition of an amino or a cyclohexylamino group to position C3 also showed no improvement of adenosine A₁ or A_2A receptor affinity. Surprisingly para-substitution on the phenyl ring at position C2 in combination with a cyclohexylamino group at position C3 led to adenosine A₁ receptor affinity in the low micromolar range with compound 4d showing: (1) the highest affinity for the adenosine A₁ receptor with a K_i value of 2.06 µM and (2) adenosine A₁ receptor antagonistic properties. This pilot study concludes that para-substituted 3-cyclohexylamino-2-phenyl-imidazo[1,2-α]pyridine analogues represent an interesting scaffold to investigate further structure-activity relationships in the design of novel imidazo[1,2-α]pyridine-based adenosine A₁ receptor antagonists for the treatment of neurodegenerative disorders.     

Cathepsin L silencing enhances arsenic trioxide mediated in vitro cytotoxicity and apoptosis in glioblastoma U87MG spheroids

Despite improved treatment options, glioblastoma multiforme (GBM) remains the most aggressive brain tumour with the shortest post-diagnostic survival. Arsenite (As₂O₃) is already being used in the treatment of acute promyelocytic leukaemia (APL), yet its effects on GBM have not been evaluated in detail. In U87MG cell monolayers, we have previously shown that arsenite cytotoxicity significantly increases upon transient inhibition of lysosomal protease Cathepsin L (CatL). As multicellular spheroids more closely represent in vivo tumours, we aimed to evaluate the impact of permanent CatL silencing on arsenite treatment in U87MG spheroids. CatL was stably silenced using shRNA expression plasmid packed lentiviruses. By using metabolic- and cell viability assays, we demonstrated that long-term CatL silencing significantly increased arsenite cytotoxicity in U87MG spheroids. Silenced CatL also increased arsenite-mediated apoptosis in spheroids via elevated p53 expression, Bax/Bcl2 ratio and caspase 3/7 activity, though with lower efficacy than in monolayers. Arsenite cytotoxicity was enhanced by lower CatL activity, since similar cytotoxicity increase was also observed using the novel CatL inhibitor AT094. The results have significant translational impact, since stable CatL silencing would enable the application of lower systemic doses of arsenite to achieve the desired cytotoxic effects on GBMs in vivo.     

Incorporation of [5-3H]uridine and attachment of cells to glass during activation of lymphocytes induced by phytohaemagglutinin

1. Shortly after the addition of phytohaemagglutinin to cultures, partially purified pig blood lymphocytes showed increased labelling of RNA by [5-(3)H]uridine and began to attach to the glass of the culture tubes. 2. In the presence of phytohaemagglutinin most cells became attached to glass during the first 5hr. of culture; those first attaching had a low mean RNA/DNA ratio. In the absence of phytohaemagglutinin cell attachment to glass was diminished. 3. During the first 5hr. of culture the rates of increase of radioactivity/unit of DNA of the attached and unattached populations in the presence of phytohaemagglutinin were of the same order and exceeded that of cultures without phytohaemagglutinin. 4. The increase of labelling in cultures to which phytohaemagglutinin was added after cells had sedimented to glass was greater than that occurring in cultures in which cells were resuspended before phytohaemagglutinin was added.     

Imperatoxin A Induces Subconductance States in Ca2+ Release Channels (Ryanodine Receptors) of Cardiac and Skeletal Muscle

Single-channel and [³H]ryanodine binding experiments were carried out to examine the effects of imperatoxin activator (IpTx_a), a 33 amino acid peptide isolated from the venom of the African scorpion Pandinus imperator, on rabbit skeletal and canine cardiac muscle Ca²⁺ release channels (CRCs). Single channel currents from purified CRCs incorporated into planar lipid bilayers were recorded in 250 mM KCl media. Addition of IpTx_a in nanomolar concentration to the cytosolic (cis) side, but not to the lumenal (trans) side, induced substates in both ryanodine receptor isoforms. The substates displayed a slightly rectifying current–voltage relationship. The chord conductance at −40 mV was ∼43% of the full conductance, whereas it was ∼28% at a holding potential of +40 mV. The substate formation by IpTx_a was voltage and concentration dependent. Analysis of voltage and concentration dependence and kinetics of substate formation suggested that IpTx_a reversibly binds to the CRC at a single site in the voltage drop across the channel. The rate constant for IpTx_a binding to the skeletal muscle CRC increased e-fold per +53 mV and the rate constant of dissociation decreased e-fold per +25 mV applied holding potential. The effective valence of the reaction leading to the substate was ∼1.5. The IpTx_a binding site was calculated to be located at ∼23% of the voltage drop from the cytosolic side. IpTx_a induced substates in the ryanodine-modified skeletal CRC and increased or reduced [³H]ryanodine binding to sarcoplasmic reticulum vesicles depending on the level of channel activation. These results suggest that IpTx_a induces subconductance states in skeletal and cardiac muscle Ca²⁺ release channels by binding to a single, cytosolically accessible site different from the ryanodine binding site.     

Interaction of fosfomycin with the Glycerol 3-phosphate Transporter of Escherichia coli

Background

Fosfomycin is widely used to treat urinary tract and pediatric gastrointestinal infections of bacteria. It is supposed that this antibiotic enters cells via two transport systems, including the bacterial Glycerol-3-phosphate Transporter (GlpT). Impaired function of GlpT is one mechanism for fosfomycin resistance.

Methods

The interaction of fosfomycin with the recombinant and purified GlpT of Escherichia coli reconstituted in liposomes has been studied. IC₅₀ and the half-saturation constant of the transporter for external fosfomycin (K_i) were determined by transport assay of [¹⁴C]glycerol-3-phosphate catalyzed by recombinant GlpT. Efficacy of fosfomycin on growth rates of GlpT defective bacteria strains transformed with recombinant GlpT was measured.

Results

Fosfomycin, externally added to the proteoliposomes, poorly inhibited the glycerol-3-phosphate/glycerol-3-phosphate antiport catalyzed by the reconstituted transporter with an IC₅₀ of 6.4 mM. A kinetic analysis revealed that the inhibition was completely competitive, that is, fosfomycin interacted with the substrate-binding site and the K_i measured was 1.65 mM. Transport assays performed with proteoliposomes containing internal fosfomycin indicate that it was not very well transported by GlpT. Complementation study, performed with GlpT defective bacteria strains, indicated that the fosfomycin resistance, beside deficiency in antibiotic transporter, could be due to other gene defects.

Conclusions

The poor transport observed in a reconstituted system together with the high value of K_i and the results of complementation study well explain the usual high dosage of this drug for the treatment of the urinary tract infections.

General significance

This is the first report regarding functional analysis of interaction between fosfomycin and GlpT.