Ferriprotoporphyrin IX and cell lysis: a protective role for hydrogen peroxide

Two potentially lytic substances, ferriprotoporphyrin IX (FP) and hydrogen peroxide, may coexist and partially detoxify each other in sickle cells and in erythrocytes infected with malaria parasites. Since hydrogen peroxide can decompose FP, its effect on hemolysis induced by FP and by the complex of FP with chloroquine was investigated. Human erythrocytes suspended at a concentration of 0.5% in a 50 microM solution of FP underwent approximately 42% hemolysis during the course of 2 hours. Twenty-five micromolar chloroquine potentiated hemolysis to 99%, and preincubation of 50 microM FP with 25 microM hydrogen peroxide for 5 minutes reduced hemolysis to 4%. Mixing either FP or hydrogen peroxide first with chloroquine abolished the effect of hydrogen peroxide. Detoxification of FP by hydrogen peroxide may be an important protective mechanism in certain hemolytic anemias, and inhibition of detoxification could account for the effectiveness of chloroquine in malaria.     

Interaction of quinoline antimalarial drugs with ferriprotoporphyrin IX, a solid state spectroscopy study

To investigate the nature of binding of quinoline antimalarial drugs to heme and to extract experimental evidence for this binding, the interaction of ferriprotoporphyrin IX (FP) with chloroquine and quinacrine (both of which have a similar side chain) and quinoline methanol antimalarials quinine and mefloquine has been studied using IR and NIR-Raman spectroscopy in the solid state. Attenuated total reflectance infrared spectroscopic data clearly show that heme in chloroquine-FP complex is not μ-oxo dimeric indicating that the hypothesis that chloroquine binds to FP μ-oxo dimer with a stoichiometry of 1 chloroquine:2 μ-oxo dimers is not valid in the solid state. Moreover, the first vibrational spectroscopy evidence is presented for the formation of hydrogen bonding between a propionate group of heme and the tertiary amino nitrogen of chloroquine and quinacrine. Raman spectroscopy data does not provide any evidence to support the formation of a similar salt bridge in the complexes of FP with quinine and mefloquine; however, it suggests that the interaction of these drugs with FP happens through coordination of the Fe(III) center of the porphyrin to the 9-hydroxy group of the drug.     

Accumulation of weak bases in relation to intralysosomal pH in cultured human skin fibroblasts

The volume of the lysosomal compartment in cultured human skin fibroblasts was estimated from the distribution between the cells and the medium of tracer amounts of labelled methylamine and chloroquine, which accumulate in the lysosomes, 2,2-dimethyloxazolidine-2,4-dione, which accumulates in the soluble cytoplasmic compartment relative to the lysosomes, and sucrose, which is excluded by the cells. In a foetal fibroblast line, the fractional volume of the lysosomal compartment was $\text{0.044 ± 0.007}$ ($\text{n}\text{= 8}$). In fibroblasts from a patient with the I-cell disease, the fractional volume was 0.15.The fractional volume of the lysosomal compartment was used to calculate the intralysosomal pH from the accumulation of the weak bases in the cells. The mean value obtained was $\text{5.29 ± 0.04}$ ($\text{n}\text{= 8}$).In fibroblasts incubated with various concentrations of chloroquine, the fractional volume of the lysosomal compartment and the accumulation of chloroquine in the cells were used to calculate the concentration of chloroquine in the lysosomes. The intralysosomal concentration increased from 3 to 114 mM as the extracellular concentration increased from 1 to 100 μM. Concomitantly, the intralysosomal pH increased from 5.3 in the absence of chloroquine to 5.9 in the presence of 100 μM chloroquine. A similar increase in intralysosomal pH could be calculated in fibroblasts incubated with different concentrations of ammonia.     

Chloroquine susceptibility in malaria: dependence on exposure of parasites to the drug.

Chloroquine-resistant $\text{P.}$$\text{berghei}$ is as susceptible to chloroquine as chloroquine-susceptible $\text{P.}$$\text{berghei}$ when adequately exposed for short periods of time (1 hour) $\text{in}$$\text{vitro}$. In both cases 3.1 mM chloroquine causes a significant decrease in infectivity of the parasites whereas 0.31 mM chloroquine is without effect. Since there is no evidence that chloroquine has a peculiar mechanism of action $\text{in}$$\text{vitro}$, these results support the hypothesis of inadequate exposure of intracellular parasites as the cause of chloroquine resistance.     

The metabolism of formycin B in Leishmaniadonovani

Formycin B, a pyrazolo(4,3-d)pyrimidine C-nucleoside, inhibited the growth of $\text{Leishmania}\text{donovani}$ promastigotes in culture with an ED₉₀ of 0.2 μg/ml. Promastigotes incubated for 24 hrs with Formycin B at 10 μg/ml were found to convert it to the ribonucleotide, formycin B 5′-monophosphate. The parasites were also capable of aminating formycin B 5′-monophosphate as evidenced by the appearance of formycin A di- and triphosphate. The RNA contained the formycin A moiety in 3′,5′-polynucleotide linkage. Succino-AMP synthetase from these parasites was able to use formycin B 5′-monophosphate as an alternate-substrate with a K'm of 26 μM and a V'm of about 1% the V'm IMP. Formycin B 5′-monophosphate was also a substrate for mammalian succino-AMP synthetase with a V_m' of 40% the V_m' of IMP.     

Chymotrypsin stimulated development of delayed implantation mouse blastocysts

Chymotrypsin-like enzyme activity increases transiently in the uterine lumen of ovariectomized mice upon administration of progesterone and estrogen (1). This is one of the few known macromolecular changes associated with conditions which result in activation of delayed implantation blastocysts $\text{in}$$\text{utero}$. $\text{In}$$\text{vitro}$, α-chymotrypsin (100 μg/ml) was found to shorten the time required for these embryos to attach to the glass culture dish and then form outgrowths in fetal calf serum-supplemented medium. Higher concentrations of the enzyme (250 μg/ml) prevented embryo attachment probably by digesting the fetuin present in fetal calf serum. Nevertheless, 250 μg/ml α-chymotrypsin could apparently replace fetal calf serum as a stimulator of development during the first 24 hours of culture. In contrast, bovine serum albumin (3.0 mg/ml) seemed to slow development of blastocysts $\text{in}$$\text{vitro}$. It is suggested that chymotrypsin-like enzyme activity may stimulate development of delayed implantation blastocysts $\text{in}$$\text{utero}$ (a) indirectly by removing inhibitory proteins such as albumin and (b) by directly affecting these embryos in a manner yet to be determined.     

Esherichia coli pyruvate dehydrogenase complex: improved purification and the flavin content.

The $\text{E. coli}$ pyruvate dehydrogenase complex, when purified by published procedures, contains phosphotransacetylase and coenzyme A as trace contaminants as well as one or more spectral contaminants which interfere with spectral and radiochemical experiments. They can be removed by further chromatographic purification on columns of calcium phosphate gel-cellulose. The resulting complexes from $\text{E. coli}$ K12 or Crookes strain are indistinguishable with respect to visible spectrum, catalytic activity, and flavin content. The activity is the highest so far reported, 40–42 μmoles DPNH per min per mg of protein, and the flavin content is 1.8–2.4 nanomoles per mg of protein.     

Effects of cytochalasin B on division and calcium carbonate extrusion in a calcareous alga.

Cytochalasin B (CB) (100 μg/ml) reversibly blocked cell division and cuased the formation of abnormal cytoplasmic bodies in the alga Cricosphaera carterae. Concentrations of 20 μg/ml and 40 μg/ml CB were without effects. In the presence of CB, calcified bodies (coccoliths) which form in Golgi vesicles and are normally extruded through the plasma membrane were not extruded and accumulated within the cell. CB appeared to alter the membranes of Golgi vesicles containing coccoliths. DMSO (10% $\text{v}\text{v}$), the solvent for CB, was without effect on cell division and coccolith extrusion. A concentration of 20% $\text{v}\text{v}$ DMSO inhibited cell division irreversibly.     

t-Butyl hydroperoxide-induced changes in the physicochemical properties of human erythrocytes

Treatment of human erythrocytes with micromolar concentrations of $\text{t-}\text{butyl}$ hydroperoxide causes a variety of changes in the physical properties of the cells. Red cells exposed to concentrations of $\text{t-}\text{butyl}$ hydroperoxide of less than 750 μM for 15 min exhibited significant decreases in cellular and membrane deformability, increases in membrane-associated protein crosslinking, osmotic fragility and the viscosity of the intracellular hemoglobin solution. No changes in the volume or density of the cells were observed. Changes in cellular deformability are probably attributable solely to changes in the mechanical properties of the cell membrane. Conversely, when red cells are exposed to $\text{t-}\text{butyl}$ hydroperoxide concentrations in excess of 750 μM for 15 min they exhibited decreases in cellular deformability which may be related to increases in cell volume as well as membrane rigidity.     

Active calcium transport in red cell ghosts resealed in dextran solutions

1.Human erythrocytes when lysed and resealed to Ca in the presence of dextran can be readily separated from the suspending medium by low-speed centrifugation. 2. Ghosts trapped Ca and EGTA at the same ratio as present in the haemolytic medium and remained tight to Ca after washing and subsequent incubation for up to 90 min at 37°C. 3. Ca extrusion could be promoted by substrates other than ATP only from ghosts that had been loaded with low free Ca concentrations (1–22 μM). The order of activation by the various substrates employed was $\text{ATP}\text{>}\text{adenine}\text{+}\text{inosine}\text{>}\text{inosine}$. 4. The kinetics of extrusion depended markedly on internal free Ca. The system showed a high affinity state ($\text{K}{}_{\mathrm{<mtext>Ca</mtext>}}\text{about}\text{3 μ}\text{M}$; $\text{V = 0.34 μ}\text{mol Ca}\text{/}\text{ml ghosts per min}$) at low concentrations (1–22 μM) and a low affinity state ($\text{K}{}_{\mathrm{<mtext>Ca</mtext>}}\text{about}\text{250 μ}\text{M}$; $\text{V = 0.17 μ}\text{mol Ca}\text{/}\text{ml ghosts per min}$) at high concentrations (0.2–4.0 mM). 5. Both at low and at high free Ca, La-sensitive ATP hydrolysis was closely correlated with La-dependent Ca efflux, in keeping with an stoichiometry of 1. 6. The rate of extrusion was maximal in the presence of 160 mM KCl and decreased to various extents when K was fully replaced by different cations, following the order $\text{K}\text{>}\text{Na = choline}\text{>}\text{Mg}$. 7. The efflux rate of high-K ghosts, resealed to alkaline cations, was stimulated by external Na, whilst Mg and choline were practically without effect. 8. The results indicate that human red cells possess a powerful Ca extrusion mechanism, the activity of which can be modulated by alkaline cations.     

Linkage of chloroquine resistance in Plasmodium berghei to infection of immature erythrocytes of mice.

Studies of infective potency were done to determine why chloroquine-resistant $\text{P.}$$\text{berghei}$ is an obligate parasite of immature erythrocytes. Infective potency was estimated from the length of time required for mice to develop parasitemia of 2% (delay in parasitemia). The delay in parasitemia was an inverse linear function of the logarithm of the number of parasites inoculated. A single regression line fitted the data both for susceptible and for resistant parasites, indicating identical infective potencies which, in turn, indicates that chloroquine-resistant $\text{P.}$$\text{berghei}$ selects and preferentially infects immature erythrocytes whereas it rejects mature erythrocytes.     

Oxygen exchange associated with electron transport and photophosphorylation in spinach thylakoids

O₂ uptake in spinach thylakoids was composed of ferredoxin-dependent and -independent components. The ferredoxin-independent component was largely 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) insensitive (60%). Light-dependent O₂ uptake was stimulated 7-fold by 70 μM ferredoxin and both uptake and evolution (with O₂ as the only electron acceptor) responded almost linearly to ferredoxin up to 40 μM. NADP⁺ reduction, however, was saturated by less than 20 μM ferredoxin. The affinity of O₂ uptake for for O₂ was highly dependent on ferredoxin concentration, with $\text{K}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{(O}{}_2\text{)}$ of less than 20 μM at 2 μM ferredoxin but greater than 60 μM O₂ with 25 μM ferredoxin. O₂ uptake could be suppressed up to 80% with saturating NADP⁺ and it approximated a competitive inhibitor of O₂ uptake with a K_i of 8–15 μM. Electron transport in these thylakoids supported high rates of photophosphorylation with NADP⁺ (600 μmol ATP/mg Chl per h) or O₂ (280 μmol/mg Chl per h) as electron acceptors, with $\text{ATP}\text{2}\text{e}$ ratios of 1.15–1.55. Variation in $\text{ATP}\text{2}\text{e}$ ratios with ferredoxin concentration and effects of antimycin A indicate that cyclic electron flow may also be occurring in this thylakoid system. Results are discussed with regard to photoreduction of O₂ as a potential source of ATP in vivo.     

Surface carbohydrates of aged erythrocytes

Human erythrocytes, fractioned into populations of different density by ultracentrifugation in albumin gradients were examined to determine what changes in cell surface carbohydrates occur during their lifespan. In addition to changes occurring in $\text{N}$-acetylneuraminic acid ageing was accompanied by reduction in the $\text{N}$-acetylglucosamine, $\text{N}$-acetylgalactosamine and galactose content of erythrocyte membranes. These results show that extensive heterogeneity exists in the cell surface carbohydrate of the circulating population of erythrocytes and suggest clearance of neuraminidase treated erythrocytes may not be an adequate model for the removal of aged cells.     

Inhibitory effect of unconjugated bilirubin on p-aminohippurate transport in rat kidney cortex slices

(1) The effects of unconjugated bilirubin on the accumulation of $\text{p-}\text{aminohippurate}$, kinetics of $\text{p-}\text{aminohippurate}$ uptake, the efflux of pre-accumulated $\text{p-}\text{aminohippurate}$ and water and electrolyte distribution were investigated in the rat kidney cortical slice. (2) The addition of unconjugated bilirubin to the incubation medium decreased the 60 min slice-to-medium concentration ratio of $\text{p-}\text{aminohippurate}$. (3) The decrease in $\text{p-}\text{aminohippurate}$ accumulation by unconjugated bilirubin was found to be more pronounced by increasing the concentration of pigment in the medium. (4) The rate of uptake of $\text{p-}\text{aminohippurate}$ as a function of $\text{p-}\text{aminohippurate}$ concentration differed in aerobiosis and anaerobiosis, and unconjugated bilirubin decreased only the uptake of $\text{p-}\text{aminohippurate}$ in aerobic conditions. (5) The efflux of pre-accumulated $\text{p-}\text{aminohippurate}$ decreased when unconjugated bilirubin concentration in the medium was low (10–20 μM) but the efflux increased when the concentration of pigment was much higher (100 μM). (6) The addition of unconjugated bilirubin to the medium (40–100 μM) increased intracellular sodium and total tissue water content, and decreased intracellular potassium and oxygen consumption of tissue. However the slices incubated with low concentration of pigment (20 μM) did not exhibit significative changes in cellular functional parameters. (7) These findings suggest that unconjugated bilirubin impairs $\text{p-}\text{aminohippurate}$ transport by a complex mechanism that might involve binding of pigment to sites necessary for anion transport, although effects related to pigment toxicity or to its oxidative decomposition are not excluded.     

Effect of adding albumin to solutions of cholecystokinin on pancreatic protein output and pancreatic polypeptide release

In four conscious dogs with chronic gastric and pancreatic Thomas fistulas we studied the effect of 99% pure cholecystokinin-33 (CCK-33) solutions on pancreatic secretion and PP release. CCK-33 was dissolved in 0.154 M NaCl alone or in the same solution containing 1 g per 100 ml dog albumin. The response of pancreatic protein output to increasing doses of CCK-33 (0.5, 1, 2, 4 IDU/kg per h) was significantly $\text{(P < 0.05)}$ higher when CCK was dissolved in NaCl with albumin than in NaCl alone. These results were confirmed by measuring CCK immunoreactivity in samples from tips of infusion lines by a gastrin radioimmunoassay. Release of pancreatic polypeptide (PP) following increasing doses of CCK-33 was also significantly $\text{(P < 0.05)}$ elevated when CCK was dissolved in an albumin-containing solution. There was a significant $\text{(P < 0.02)}$ correlation between plasma concentrations of PP and pancreatic protein output.This study suggests that albumin should be added to CCK-33 solutions to preserve biological activity. The biological effect of CCK-33 may be substantially underestimated if albumin is omitted.     

Malaria enhances cyclic AMP production by immature erythrocytes in vitro.

Infection with a chloroquine-susceptible line of $\text{Plasmodium}$$\text{berghei}$ NYU-2 enhanced the production of cyclic AMP in response to isoproterenol only in immature mouse erythrocytes, which possess a functional hormone-receptor-adenylate cyclase complex. With 10^?7 M isoproterenol the increases in cyclic AMP concentrations (picomoles per 10⁸ erythrocytes) were 19 for a preparation of mature erythrocytes, 81 for a preparation enriched with immature erythrocytes, 25 for a preparation of infected mature erythrocytes, and 900 for a preparation enriched with infected immature erythrocytes. Beta-adrenergic receptor blockade with propranolol prevented this response to isoproterenol but had no effect on the stimulation produced by prostaglandin E₁. These findings indicate that the malaria parasite enhances the responsiveness of a fundamental regulatory system that is intrinsic to immature erythrocytes.     

Further studies on the pharmacology of a false cholinergic transmitter, (2-hydroxyethyl) methyldiethylammonium (diethylcholine).

The pharmacology of a possible false cholinergic transmitter, (2-hydroxyethyl) methyldiethylammonium (diethylcholine, DEC) was studied with various preparations. It was found to inhibit the neuromuscular transmission of frog sciatic nerve-gastrocnemius muscle $\text{in}$$\text{vitro}$ with ED₅₀ of 1.93 (0.66 - 5.79) × 10⁻⁴ M. DEC was also found to inhibit dog chorda tympani-Wharton's duct (postganglionic parasympathetic neuro-effector junction) and cat superior cervical ganglionnictitating membrane (sympathetic ganglion) preparations $\text{in}$$\text{vivo}$ with ED₅₀'s of 6.2 (1.8 – 21.1) mg/kg and 12.0 (5.7 - 25.2) mg/kg, respectively. After blockade of these preparations with DEC, the former was still responsive to intravenous injection of pilocarpine (1 mg/kg) and choline (10 mg/kg) and the latter to close arterial injection of acetylcholine (100 μg/injection) and choline (3 mg/min infusion). These results support the idea that DEC paralyzes cholinergic neurons possibly through false cholinergic transmission without blocking the cholinergic receptor at the post-junctional membrane.     

Diacytosis of 125I-asialoorosomucoid by rat hepatocytesa. A non-lysosomal pathway insensitive to inhibition by inhibitors of ligand degradation

Diacytosis of ¹²⁵I-asialoorosomucoid by rat hepatocytes was studied by preincubating the cells with the labelled ligand at 37°C for 30 min or 18°C for 2 h, washing free of cell surface receptor-bound tracer at 4°C and then reincubating at 37°C. The cells preloaded at 37°C released a maximum of 18% of the total intracellular ligand as undegraded molecules after 1 h of incubation with an apparent first-order rate constant of 0.018 min^?1 ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 39}\text{min}$). When the preloaded cells were incubated in the presence of 100 μg/ml unlabelled asialoorosomucoid or 5 mM ethylene glycol bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid, the amount of the released ligand increased to 32 and 37%, respectively, without apparent change in kinetics, indicating that these agents prevented rebinding of the released ligand. In the presence of 5 μM colchicine, 20 μM cytochalasin B, 20 μM chloroquine, 10 mM NH₄Cl, 10 μM monensin or 20 μM leupeptin, degradation of the preloaded ligand was inhibited, whereas the release of the ligand was either slightly increased or unchanged. Similar effects of leupeptin, colchicine and asialoocrosomucoid were observed with cells preloaded at 18°C. These results indicate that diacytosis of ¹²⁵I-asialoorosomucoid occurs from a prelysosomal compartment via a route insensitive to inhibition by the inhibitors of ligand degradation.     

Synthesis and antitumor activity of 1-β-D-arabinofuranosylcytosine-5′-diphosphate-prednisolone and -cortisol

Superior antitumor activity and reduced toxicity of 1-β-arabinofuranosylcytosine (ara-C) conjugates of corticosteroids through a phosphodiester linkage have prompted us to synthesize the new ara-C conjugates of corticosteroids through a pyrophosphate diester linkage. Condensation of ara-CMP morpholidate with the steroid-21-monophosphates in pyridine at room temperature for 6 days and the subsequent separation on a DE-52 (formate) column using a linear gradient of 0?0.5 M triethylammonium formate (pH 7.0) gave ara-CDP-prednisolone (I) and ara-CDP-cortisol (II) in 37–55% yield. The conjugates were hydrolyzed to ara-CMP and the corresponding steroid-21-monophosphate by phosphodiesterase I. Conjugates I and II inhibited the $\text{in}\text{,}\text{vitro}$ growth of L1210 lymphoid leukemia by 50% (ED₅₀) at 0.03 and 0.08 μM, respectively, while ara-C and ara-CMP showed the respective ED₅₀ of 0.1 μM and 0.05 μM. Conjugates I and II exhibited ILS values of 116 and 86%, respectively, at 40 mg (53.7 μmole)/kg/day × 5 against L1210 leukemic mice, while that of ara-C at the nearly same dose (49 μmole/kg/day × 5) was 65%.