High Cellular Concentrations Research Articles

Les nouveaux macrolides

Following their use for a few years, the place of new macrolides can be assessed."New macrolides", ie, roxithromycin, clarithromycin, azithromycin and dirithromycin, are derivated from erythromycin and defined by different pharmacokinetic parameters: longer half time with better oral administration and shorter duration of treatment, better tissue concentrations with reduction of dosages and better tolerance, high cellular concentration with good activity. However, new macrolides and erythromycin do not show major differences in their antibacterial spectrum against usual pyogenic strains. Clinical use of new macrolides extends to opportunistic infections, such as atypical mycobacterial infections or toxoplasmosis, occurring in the course of HIV infection. New macrolides are also recommended for the treatment of Helicobacter pylori-related gastroduodenal ulcer.New macrolides have opened new avenues in the development of anti-infectious strategies. Due to their good efficacy against Chlamydia pneumoniae which is suspected of inducing vascular diseases, the upcoming use of ketolids and the development of their non-antibiotic effects, the future of macrolides is favorable.

The glutathione redox couple modulates zinc transfer from metallothionein to zinc-depleted sorbitol dehydrogenase.

The release and transfer of zinc from metallothionein (MT) to zinc-depleted sorbitol dehydrogenase (EC 1.1.1.14) in vitro has been used to explore the role of MT in cellular zinc distribution. A 1:1 molar ratio of MT to sorbitol dehydrogenase is required for full reactivation, indicating that only one of the seven zinc atoms of MT is transferred in this process. Reduced glutathione (GSH) and glutathione disulfide (GSSG) are critical modulators of both the rate of zinc transfer and the ultimate number of zinc atoms transferred. GSSG increases the rate of zinc transfer 3-fold, and its concentration is the major determinant for efficient zinc transfer. GSH has a dual function. In the absence of GSSG, it inhibits zinc transfer from MT, indicating that MT is in a latent state under the relatively high cellular concentrations of GSH. In addition, it primes MT for the reaction with GSSG by enhancing the rate of zinc transfer 10-fold and by increasing the number of zinc atoms transferred to four. 65Zn-labeling experiments confirm the release of one zinc from MT in the absence of glutathione and the more effective release of zinc in the presence of GSH and GSSG. In vivo, MT may keep the cellular concentrations of free zinc very low and, acting as a temporary cellular reservoir, release zinc in a process that is dynamically controlled by its interactions with both GSH and GSSG. These results suggest that a change of the redox state of the cell could serve as a driving force and signal for zinc distribution from MT.

Cell- and tissue-specific metabolic activation of chemicals as determined by autoradiography: in vitro—in vivo correlations

Numerous chemicals require metabolic activation to exert toxicity. Such metabolism-dependent toxicity may be due to the formation of electrophiles that bind irreversibly to cellular macromolecules in the vicinity of their site of formation. A high cellular concentration of a covalently bound metabolite may thus imply a target site for toxicity. Autoradiography at different levels of resolution is useful to determine cell-specific covalent metabolite binding of radiolabelled chemicals, both in vivo and in vitro. Using this approach, cell-specific toxicants such as dichlobenil (olfactory mucosa) and MeSO 2-DDE (adrenal cortex) have been identified. In addition, unforeseen sites of local metabolism of toxic chemicals have been demonstrated. In our experience, the in vitro-in vivo correlations are often good, while in some cases results obtained in vivo cannot be reproduced in vitro. The inability of tissue slices to account for in vivo events may depend on metabolism in non-target tissues and differences in tissue disposition in vivo and in vitro. Examples are given to demonstrate the utility of autoradiography in vitro and in vivo to trace metabolism-dependent binding and toxicity in airway epithelia, vascular endothelia and adrenal zona fasciculata cells.

Copper-binding motifs in catalysis, transport, detoxification and signaling.

Copper is required for many biological processes but is toxic at high cellular concentrations, so levels in the cell must be strictly controlled. Copper-binding motifs have been identified and characterized in many proteins. The way in which copper is coordinated by these motifs is important for the transport and distribution of intracellular copper and for the effective functioning of copper-dependent enzymes.

Effects of Dithiocarbamate and 8-Hydroxyquinoline Additions on Algal Uptake of Ambient Copper and Nickel in South San Francisco Bay Water

The addition of synthetic organic ligands such as diethyldithiocarbamate (DDC) and 8-hydroxyquinoline (Ox) to the dissolved fraction (<0.2 μm) of South San Francisco Bay water facilitated the transport of ambient Cu and Ni into phytoplankton cells. The uptake mechanism is diffusion of the lipophilic organic Cu(DDC)2 0 and Cu(Ox)2 0 complexes (and corresponding Ni complexes) across the plasma membrane. Short-term uptake experiments were carried out using a coastal diatom,Thalassiosira weissflogii, and resulted in rapid uptake rates and high cellular concentrations of the metal relative to the bay water control. Steady-state conditions between the solution and cellular Cu concentrations occurred within 10 min for a 4 μM addition of DDC and after 3 h for a 100 μM addition of Ox. Steady-state cellular Cu concentrations were over 10 times and 6 times greater, for DDC and Ox treatments, than in the bay water controls. Steady-state cellular Ni concentrations were attained within 10 min for both ligand additions and were more than 6 times and 2 times greater than in the absence of the added ligands. Using this bioassay, we were also able to gain insight into the character of the background organic Cu complexes in South Bay. Our results suggest that the natural organic Cu complexes are relatively hydrophilic in character and do not appear to be directly assimilated across the plasmalemma.

Antagonisms between cadmium and zinc toxicity and manganese limitation in a coastal diatom

Long‐term growth and metal (Mn and Cd) uptake experiments and short‐term uptake kinetic experiments with the coastal diatom Thalassiosira pseudonana revealed antagonistic interactions between toxic metals (Zn and Cd) and the micronutrient manganese. Cd and Zn inhibited algal growth rate only at low Mn ion concentrations, an effect that could be totally accounted for by an inhibition of cellular Mn uptake by the toxic metals and the resultant generation of Mn‐deficient cells. Mn and Zn inhibited cellular Cd uptake, indicating reciprocal effects among the metals with respect to uptake. Saturation kinetics modeling of the uptake data was consistent with all three metals competing with each other for binding to the Mn uptake system and with both Cd and Mn being transported into the cell by that system (uptake of Zn was not measured). Mathematical modeling of Mn and Cd uptake data revealed evidence for a Cd efflux system that was induced only at high cellular Cd concentrations and prevented cellular Cd from reaching toxic levels.

High cellular concentration of peripheral blood progenitor cells during cryopreservation adversely affects CFU-GM but not hematopoietic recovery.

The effects of bone marrow (BM) and peripheral blood progenitor (PBPC) concentration during cryopreservation on subsequent hematopoietic engraftment following high-dose chemotherapy were studied in 24 patients. Seventeen BM harvests and 71 PBPC collections were performed between July 1991 and June 1994. The PBPC were frozen at significantly higher cellular concentrations than the BM (medians of 243 x 10(6)/ml versus 73 x 10(6)/ml respectively, p = 0.0003). The recovery of committed progenitor cell colonies (CFU-GM) was significantly lower from PBPC frozen at concentrations above the median, compared with 116% from those frozen at concentrations below the median (p = 0.0467). This phenomenon was not seen in BM, which was generally frozen at a threefold lower concentrations. Despite the lower recovery of CFU-GM when PBPC were frozen at a higher concentration, the patients receiving these grafts achieved good hematopoietic recovery. The higher number of PBPC probably compensated for the loss, and the patients still received a substantial number of clonogenic hematopoietic precursors.

Does P-Glycoprotein Play a Pivotal Role in the Drug Resistance of an MDR Variant, K562/Dox?

A multidrug-resistant (MDR) variant, K562/Dox, was selected from repeated exposure of human erythroleukemia cell line K562 to doxorubicin (Dox). K562/Dox displayed typical MDR features with respect to its cross-resistance to a variety of functionally and structurally unrelated compounds: vincristine (Vin), Dox, mitomycin C, reduced steady-state intracellular anthracycline accumulation, and elevated P-glycoprotein expression/mdr1 mRNA transcription/mdr1 gene amplification. Nevertheless, by incubation of cells with Dox/epirubicin (Epi)/daunorubicin (Dau) (5-80 micrograms/ml), the initial drug uptake was similar (p > 0.05) in K562/Dox and K562 cells, suggesting P-glycoprotein-mediated drug efflux would not occur unless a relatively high cellular drug concentration was reached. After 8 h incubation of cells with 50 ng/ml Dox (5 times higher than its IC50 to K562 cells), there were only slight differences (p > 0.05) in intracellular drug levels between K562/Dox and K562 cells, clearly indicating that K562/Dox, circumventing drug toxicity in this case, was irrelevant to reduced drug accumulation caused by P-glycoprotein. Similar results were obtained when Epi or Dau was applied. Despite complete restoration of anthracycline accumulation in K562/Dox cells in the presence of 6 mumol/l verapamil, the reversal of their drug resistance was incomplete. These results suggest that P-glycoprotein-mediated drug efflux possibly did not play a primary role in the drug resistance of K562/Dox cells.

Chapter 23 Assaying Proteins for Molecular Chaperone Activity

Publisher Summary This chapter discusses the assaying proteins for molecular chaperone activity. Molecular chaperones play critical roles in protein folding, assembly, and translocation. Additionally, molecular chaperones maintain other proteins in translocationally competent, unfolded conformations and also function during subsequent folding. Recent evidence has suggested that molecular chaperones are probably the cellular factors responsible for this discrepancy. The cellular requirement for chaperones is because of the adverse conditions that can exist in cells that jeopardize productive protein folding. These conditions include the high cellular concentration of folding intermediates that are prone to aggregation or misfolding or that can result from the presence of environmental stresses—such as heat shock—which promote protein unfolding. Several properties define molecular chaperone activity: (1) the ability to recognize and bind unfolded proteins, (2) to suppress aggregation during protein unfolding and folding, (3) to influence the yield of folding, and (4) to carry out properties 2 and 3 at approximately stoichiometric levels.

Catalysis of protein folding by cyclophilins from different species

Cyclophilins are a class of ubiquitous proteins with yet unknown function. They were originally discovered as the major binding proteins for the immunosuppressant cyclosporin A. The only known catalytic function of these proteins in vitro is the cis/trans isomerization of Xaa-Pro bonds in oligopeptides. This became clear after the discovery that bovine cyclophilin is identical with porcine prolyl isomerase. This enzyme accelerates slow, proline-limited steps in the refolding of several proteins. Here we demonstrate that the cyclophilins from man, pig, Neurospora crassa, Saccharomyces cerevisiae, and Escherichia coli are all active as prolyl isomerases and as catalysts of protein folding. This evolutionary conservation suggests that catalysis of prolyl peptide bond isomerization may be an important function of the cyclophilins. It could be related with de novo protein folding or be involved in regulatory processes. Catalysis of folding is very efficient in the presence of the high cellular concentrations of prolyl isomerase.

The effect of ara-C-induced inhibition of DNA synthesis on its cellular pharmacology

The cytotoxicity of ara-C is believed to result from incorporation of ara-CTP into DNA and inhibition of DNA synthesis. Since complete inhibition of DNA synthesis would prevent further incorporation of ara-CTP, ara-C may have a self-limiting effect on its own cytotoxicity, particularly at the high concentrations typical of high-dose ara-C clinical protocols. In this study, the incorporation of [3H]-dThd and [3H]-ara-C into DNA were compared. Within 1 h of exposure of L5178Y cells to ara-C, the rate of [3H]-dThd incorporation into the acid-insoluble fraction was reduced by 98%. Despite this nearly complete block in [3H]-dThd incorporation, DNA synthesis was not completely inhibited since [3H]-ara-C continued to be incorporated for up to 6 h, although a plateau in ara-CDNA synthesis was observed between 2 and 3 h exposure when ara-CTP levels were maximal. The effect of ara-C on [3H]-dThd incorporation into DNA was due in part to an indirect effect of ara-C on the metabolism of intracellular [3H]-dThd to [3H]-dTTP. Within 30 min exposure to 10 microM ara-C, the rate of cellular [3H]-dTTP synthesis was slowed to only 15% of the control rate. This was not due to inhibition of [3H]-dThd transport, since the intracellular and extracellular concentrations of the nucleoside were equal. The effect of ara-C on [3H]-dTTP synthesis resulted from significant changes in deoxynucleoside 5'-triphosphate (dNTP) pools. dTTP, dATP, and dGTP levels were increased, whereas the dCTP concentration was decreased. When dThd kinase from L5178Y cells was assayed with increased dTTP levels induced by ara-C vs the dTTP level in control cells, its activity was reduced by 72%. Thus, the [3H]-dThd incorporation experiment overestimated the extent of inhibition of DNA synthesis by ara-C due to increased feedback inhibition of dThd kinase and increased competition for DNA polymerase between the elevated unlabeled dTTP pool and the decreased levels of [3H]-dTTP. In vitro assay of DNA polymerase in the presence of the ara-CTP concentration achieved after 0.5 or 3 h exposure to 10 microM ara-C (60 microM and 200 microM, respectively), plus the mixture of dNTPs found intracellularly at these times, resulted in 57% and 80% inhibition of the polymerase, respectively. This inhibition may account for the plateau in the accumulation of ara-CDNA that was observed at 3 h and suggests that ara-C incorporation may be self-limiting at high cellular concentrations of ara-CTP.(ABSTRACT TRUNCATED AT 400 WORDS)

Lead-dithiocarbamate interaction

Dithiocarbamates are known to markedly increase tissue uptake of lead and potentiate toxic effects of lead in rats. Effects of the interaction between lead and diethyldithiocarbamate (DTC) on the enzyme delta-aminolevulinic acid dehydratase, ALAD, were studied in primary cultures of rat hepatocytes, incubated with lead acetate, PbAc, or lead-diethyldithiocarbamate complex, Pb(DTC)2, labeled with 203Pb. Incubation of cells with the lipophilic Pb(DTC)2 complex caused a more rapid and stronger inhibition of ALAD activity than did PbAc. The effects of ALAD activity could be related to the cellular uptake of lead. Thus, a much higher and more rapid cellular uptake of lead was found following incubation with Pb(DTC)2 than with PbAc. Pb(DTC)2 inhibited ALAD activity also in vitro when incubated with purified ALAD enzyme. It is suggested that the increased inhibition of ALAD activity by Pb(DTC)2 is owing to facilitated cellular transport of lead in the complexed form and that complexing lead with DTC could serve as a experimental model in order to obtain high cellular concentrations of lead.

The structure and biosynthesis of new tetrahydropyrimidine derivatives in actinomycin D producer Streptomyces parvulus. Use of 13C- and 15N-labeled L-glutamate and 13C and 15N NMR spectroscopy.

Two novel compounds, 2-methyl, 4-carboxy, 5-hydroxy-3,4,5,6-tetrahydropyrimidine (THP(A] and 2-methyl, 4-carboxy-3,4,5,6-tetrahydropyrimidine (THP(B] have been identified in the pool of Streptomyces parvulus by in vivo and in vitro studies. 13C and 15N were introduced into the compounds by feeding S. parvulus with 15N- and 13C-labeled L-glutamate. High resolution 13C and 15N NMR have been applied to elucidate their structure and biosynthesis in S. parvulus. The splitting patterns and coupling constants of adjacent nitrogen-carbon molecular fragments enable us to unravel their molecular structure. Two different glutamate pools are responsible for their biosynthesis, THP(A) carbon skeleton derives from the extracellular L-[13C]glutamate, whereas THP(B) stems from D-fructose via the intracellular glutamate. During cell growth, THP(A) is synthesized and becomes the major constituent of the intracellular pool. It is consumed after THP(B) is accumulated intracellularly. The onset of THP(A) and -(B) synthesis seems correlated to the time of actinomycin D synthesis. Their high cellular concentrations during actinomycin D synthesis suggest that they may function as nitrogen storage. Other possible functions of THP molecules within the cell are discussed.

Photoinactivation and long-term photosensitivity of diploid and disomic strains of the smut fungusUstilago violacea

Diploid and disomic strains ofUstilago violacea, containing high cellular concentrations of cytochrome c and various cellular concentrations of carotenes, were subjected to high levels of radiation from incandescent, fluorescent, and UV sources to characterize inactivation kinetics. Additionally, these strains were grown under high and low levels of radiation from the fluorescent and incandescent sources, to characterize their long-term resistance to photoinactivation by visible light with different spectral qualities. Survival kinetics in response to incandescent radiation were characterized by either an exponential decay for the low carotene strain, or an initial loss of viability, with recovery and a subsequent gradual loss of viability for the strains containing high carotene concentrations. Long-term colony survival was suppressed at the high incandescent level for the low carotene strain only. Long-term incandescent exposure did not affect the other strains. Survival kinetics in response to fluorescent and UV radiation were characterized as exponential decays for all strains, however, the exposure time leading to 90% loss in viability LD90 was quantitatively related to carotene content in the form of a power function, where LD90 increased as the total carotene content per cell. We also determined that the ratio of colored carotenes to cytochrome c in the cells was quantitatively related to the rate of viability loss during UV exposure. In all cases, including the long-term fluorescent experiment, the strains containing higher concentrations of carotenes were more resistant to destruction by the different radiation sources.

Effects on Casuarina and Allocasuarina Species of Increasing Sodium Chloride Concentrations in Solution Culture

Ten species of Casuarina and Allocasuarina were exposed to increasing levels of NaCl (max 550 mM) in solution culture over a period of 5 months. Na+ and Cl- ion concentrations increased in the tissues of all species as the salinity increased and K+ was selectively accumulated. Those species which ultimately survived to the highest salinities tested had lower concentrations of Na+ and Cl- in both shoot and roots and lower Na+/K+ ratios than the more sensitive species at low external salinities. Osmotic adjustment of the Casuarina species was principally accounted for by Na+ and Cl- in the shoots. Turgor pressure was not lost until the plants were close to death, so that inhibition of height growth by NaCl was probably due to the high cellular ion concentrations. Plants were more sensitive to osmotic shock than to a gradual increase in salinity.

Control of sodium permeability of the outer barrier in toad skin.

The 24Na efflux (JNaeff) (i.e., the rate of appearance of 24Na in the outer compartment) in the isolated short-circuited toad skin bathed by NaCl-Ringer's solution on both sides is composed of para- and transcellular components of almost equal magnitudes. This relies on the assumption that amiloride acts on the transcellular component only and could block it completely. Ouabain induces a large transient increase of the transcellular component. This increase, which starts within a few minutes after the addition of ouabain, is due to electrical depolarization of the outer barrier, rather than a consequence of blocking Na recirculation across the inner barrier. The subsequent decline of JNaeff, which takes place after the ouabain-induced JNaeff peak, is due to a progressive block of outer barrier Na channels with time, which can eventually be complete, depending on the duration of action of ouabain. As the external Na concentration was always kept high and constant in these experiments, the results indicate that a rise in cell Na concentration, and not in the outer bathing solution, is the signal that triggers the reduction of outer barrier Na permeability (PNao). Ouabain has no effect upon JNaeff with Na-free solution bathing the outer and NaCl-Ringer's solution the inner skin surface, showing the importance of Na penetration across the outer barrier, and not across the inner barrier due to its low Na permeability, in the process of closing the Na channels of this structure. Step changes from Na 115 mM to Na-free external solution, or vice-versa, may affect both the outer barrier electrical potential difference (PDo) and cell Na concentration (Na)c. Therefore, the behavior of JNaeff depends on which variable (if PDo or (Na)c regulated outer barrier Na permeability) is most affected by step changes in outer bathing solution Na concentration. Amiloride in the control condition blocks the transcellular component of JNaeff. However, in the condition of approximate short-circuiting of the outer barrier and high cellular Na concentration induced by long term effects of ouabain, when the Na channels of the outer barrier are already blocked by elevated cell Na concentration, amiloride may induce the opposite effect, increasing Na permeability of the outer barrier. With outer barrier Na channels completely blocked by high cell Na concentration, PCMB in the outer bathing medium induces a large increase of JNaeff, rendering these channels again amiloride sensitive. The results are consistent with the notion that Na efflux from cell compartment to the outer bathing solution goes through the amiloride-sensitive Na channels of the apical border of the superficial cell layer of toad skin, with an apparent Na permeability modulated by cell ionic environment, most probably the cell Na concentration.

Removal of PHA from supernatants containing T-cell growth factor

Supernatants of human PBL cultures stimulated with PHA contain a lymphokine named T-cell growth factor (TCGF). To remove the contaminant PHA from these supernatants, we have designed a simple and effective method based on the binding properties of this lectin to chicken red blood cells. This procedure completely removes the functional activities of a PHA solution, both in lectin-induced cellular cytotoxicity against erythroid targets and in DNA synthesis of fresh PBL. The TCGF activity remaining in the supernatants after absorption with CRBC is dependent on the cellular concentration employed in the preparation of the supernatants. The absorption procedure does not remove significant TCGF activity in the supernatants prepared at high cellular concentrations (5 × 10 6 cells/ml), whereas in those prepared at low cellular concentrations (1 × 10 6 cells/ml) a partial loss of TCGF activity is detected.

Algal response to copper under riverine conditions

Algal species isolated from Furnace Brook, Stafford, Connecticut, USA, which received a copper-containing effluent, were culturèd for periods of about a week in the river throughout a yearly cycle. The algae were contained in dialysis tubing tied at both ends which allowed free exchange of dissolved substances between the river and the algae. At the end of the incubation period the algae were counted, harvested by filtration and analysed for copper content. The growth rates ranged from 0 to 1·12 doublings a day in sections of the river polluted by copper and were highest in the summer despite highest concentrations of dissolved copper during that time (135–687 μg litre −1). Accumulation of copper by the algae reached 2·0 × 10 −7 μg per cell. Algal growth rates were correlated with temperature in all but one case, and were not correlated with the dissolved copper concentration of the water. In laboratory experiments with increased ionic copper concentrations in the medium growth rates decreased less at the optimal temperature than at other temperatures. Decreased toxicity at optimal temperatures occureed despite relatively high cellular concentrations of copper.

The relA gene is not required for glycogen accumulation during NH 4+ starvation of Escherichia coli

When glucose is the carbon source, glycogen accumulates in partial (NH 4 +) or total (NH 4 + and required amino acids) nitrogen starvation in Escherichia coli strains that are relA + or relA −. But glycogen accumulates only in the relA + strain when required amino acids are depleted or isoleucine starvation is induced by valine addition. However, when glycerol is the carbon source, glycogen accumulates in both relA + and relA − strains after valine addition. We conclude that the relA gene is not required for glycogen accumulation when synthesis of all nitrogen-containing compounds of the cell is limited or abolished. We also conclude that, although the relA gene is needed for glycogen to accumulate in amino acid starvation, this requirement can be replaced by a high cellular concentration of 3′,5′-cyclic AMP.

Starvation-induced changes in transport of ketone bodies across the blood-brain barrier.

The permeability of the blood brain barrier (BBB) to beta-hydroxybutyrate (beta-HB) was computed in fed and starved (five days) rats by the simultaneous measurement of cerebral blood flow (diffusible indicator method-123I-iodoantipyrine) and brain uptake of 14C-beta-HB (relative to a 3H2O reference). The results from the present study demonstrate that the movement of beta-HB across the BBB in rat is by a carrier-mediated process. During starvation, total movement (carrier-mediated and diffusionary) of this ketone body into brain was observed to be enhanced because of an increase in the diffusionary loss across the cerebral capillary. The calculated transport kinetics also suggest that the beta-HB molecule has a greater affinity for the transport (mediator) protein during starvation, although the maximal rate of uptake by brain due to a carrier processes mediated Vmax is decreased either because there is a smaller quantity of the mediating molecule or because there is trans inhibition by a high cellular concentration of beta-HB or some analog.