Photodynamic activity of a new sensitizer derived from porphyrin-C60 dyad and its biological consequences in a human carcinoma cell line

The photokilling activity of a porphyrin-C₆₀ (P-C₆₀) dyad was evaluated on a Hep-2 human larynx-carcinoma cell line. This study represents the first evaluation of a dyad, with high capacity to form a photoinduced charge-separated state, to act as agent to inactivate cells by photodynamic therapy (PDT). Cell treatment was carried out with 1 μM P-C₆₀ incorporated into liposomal vesicles. No dark cytotoxicity was observed using 1 μM P-C₆₀ concentration and during long incubation time (24 h). The uptake of sensitizer into Hep-2 was studied at different times of incubation. Under these conditions, a value of 1.5 nmol/10⁶ cells was found after 4 h of incubation showing practically no change even after 24 h. The cell survival after irradiation of the cells with visible light was dependent upon light exposure level. A high photocytotoxic effect was observed for P-C₆₀, which inactivated 80% of the cells after 54 J/cm² of irradiation. Moreover, the dyad kept a high photoactivity even under argon atmosphere. Thus, depending on the microenviroment where the sensitizer is localized, this compound could produce a biological photodamage through either a ¹O₂-mediated photoreaction process or a free radical mechanism under low oxygen concentration.

The mechanism of cell death was analyzed by Hoechst-33258, toluidine blue staining, TUNEL and DNA fragmentation. Cell cultures treated for 24 h with P-C₆₀ and irradiated with a dose of 54 J/cm² showed a great amount of apoptotic cells (58%). Moreover, changes in cell morphology were analyzed using fluorescence microscopy with Hoechst-33258 under low oxygen concentration. Under this anaerobic condition, necrotic cellular death predominated on apoptotic pathway. There were more apoptotic cells under air irradiation condition than under argon irradiation condition. To determine the apoptotic pathway, caspase-3 activation was studied by caspase-3 activity detection kits. The last results showed that P-C₆₀ induced apoptosis by caspase-3-dependent pathway. These results indicated that molecular dyad, which can form a photoinduced charge-separated state, is a promising model for phototherapeutic agents and they have potential application in cell inactivation by PDT.     

Hypericin and photodynamic treatment do not interfere with transport of vitamin C during respiratory burst

Hypericin is a photosensitizing pigment found in St. John's wort (Hypericum perforatum) displaying a high toxicity towards certain tumors. The fact that some non-tumor cells, especially monocytes and granulocytes, are resistant to its photocytotoxic effects, posed the question whether this insensitivity is due to their ability to accumulate vitamin C, an antioxidant which alleviates the deleterious work of free radicals.

HL-60 promyelocytic tumor cells can be differentiated to neutrophilic granulocytes by treatment with dimethylsulfoxide and were used as cell model. In the differentiated cells, treatment with phorbol esters (PMA) stimulates vitamin C (ascorbate) transport. The uptake rates were unaltered by hypericin at concentrations below 1 μM and irradiation with visible light at a light dose of 6 J/cm². Inhibition by higher concentrations of hypericin was most probably due to a combination of photocytotoxic properties of the dye and oxygen radicals generated during respiratory burst. Superoxide production by NADPH oxidase followed by reduction of ferricytochrome c was inhibited by hypericin. The degree of inhibition was dependent on the concentration of hypericin and light intensity: IC₅₀-values were 1.7 and 0.7 μM under light doses of 3.6 and 10.8 J/cm², respectively. Oxidative stress, monitored with 2',7'-dichlorofluorescein (DCF) was only slightly decreased by ascorbate even at higher concentrations of hypericin. In contrast to its effect on the ferricytochrome c-reduction, irradiation had no significant influence on DCF-fluorescence. However, the viability of the cells was strongly decreased after photosensitization and no significant improvement was obtained by ascorbate.

Results from this work indicate that ascorbate transport per se is not altered during photodynamic therapy and vitamin C does not interfere with hypericin-induced photodamage of cellular targets.     

Depletion of intracellular Ca2+ store itself may be a major factor in thapsigargin-induced ER stress and apoptosis in PC12 cells

The mechanisms of intracellular calcium store depletion and store-related Ca²⁺ dysregulation in relation to apoptotic cell death in PC12 cells were investigated at physiological temperatures with a leak-resistant fluorescent indicator dye Fura-PE3/AM by a cooled CCD imaging analysis system. Electron microscopic observations have shown thapsigargin (TG; 100 nM)-induced apoptosis in PC12 cells. Thorough starvation of stored Ca²⁺ by BAPTA/AM (50 μM), or La³⁺ (100 μM) enhanced while dantrolene (100 μM) attenuated the TG-induced apoptosis by preventing a calcium release from internal stores. An immunoblotting analysis revealed an enhanced expression of GRP78, the hallmark of endoplasmic reticulum (ER) stress when cells were treated by TG along with BAPTA/AM. These results indicate that the depletion of the intracellular Ca²⁺ stores itself induces the ER stress and apoptosis in PC12 cells without any involvement of the capacitative calcium entry (CCE) or a sustained elevation of intracellular Ca²⁺ concentrations ([Ca²⁺]_i).     

Effects of peptide YY and its analogues on chloride ion secretion in fed and fasted rat jejunum

The aim of our study was to determine whether a meal modifies the antisecretory response induced by PYY and the structural requirements to elicit antisecretory effects of analogue PYY(22–36) for potential antidiarrhea therapy. The variations in short-circuit current (Isc) due to the modification of ionic transport across the rat intestine were assessed in vitro, using Ussing chambers. In fasted rats, PYY induced a dose- and time-dependent reduction in Isc, with a sensitivity threshold at 5 × 10⁻¹¹ M (ΔIsc −2 ± 0.5 μA/cm²). The reduction was maximal at 10⁻⁷ M (Isc −23 ± 2 μA/cm²), and the concentration producing half-maximal inhibition was 10⁻⁹ M. At 10⁻⁷ M, reduction of Isc by PYY reached 90% of response to 5 × 10⁻⁵ M bumetanide. The PYY effect was partly reversed by 10⁻⁵ M forskolin (Isc +13.43 ± 2.91 μA/h·cm², p < 0.05) or 10⁻³ M dibutyryl adenosine 3′,5′ cyclic monophosphate (Isc +12 ± 1.69 μA/cm², p < 0.05). Naloxone and tetrodotoxin did not alter the effect of PYY. In addition, PYY and its analogue P915 reduced net chloride ion secretion to 2.85 and 2.29 μEq/cm² (p < 0.05), respectively. The antisecretory effect of PYY was accompanied by dose- and time-dependent desensitization when jejunum was prestimulated by a lower dose of peptide. The antisecretory potencies exhibited by PYY analogues required both a C-terminal fragment (22–36) and an aromatic amino acid residue (Trp or Phe) at position 27. At 10⁻⁷ M the biological activity of PYY was lower in fed than fasted rats (p < 0.001). Our results confirm the antisecretory effect of PYY, but show that the fed period is accompanied by desensitization, similar to the transient desensitization observed in the fasted period with cumulative doses. This suggests that PYY may act as a physiological mediator that reduces intestinal secretion.     

Effect of clomiphene on Ca movement in human prostate cancer cells

The effect of clomiphene, an ovulation-inducing agent, on cytosolic free Ca²⁺ levels ([Ca²⁺]_i) in populations of PC3 human prostate cancer cells was explored by using fura-2 as a Ca²⁺ indicator. Clomiphene at concentrations between 10-50 μM increased [Ca²⁺]_i in a concentration-dependent manner. The [Ca²⁺]_i signal was biphasic with an initial rise and a slow decay. Ca²⁺ removal inhibited the Ca²⁺ signal by 41%. Adding 3 mM Ca²⁺ increased [Ca²⁺]_i in cells pretreated with clomiphene in Ca²⁺-free medium, confirming that clomiphene induced Ca²⁺ entry. In Ca²⁺-free medium, pretreatment with 50 μM brefeldin A (to permeabilize the Golgi complex), 1 μM thapsigargin (to inhibit the endoplasmic reticulum Ca²⁺ pump), and 2 μM carbonylcyanide m-chlorophenylhydrazone (to uncouple mitochondria) inhibited 25% of 50 μM clomiphene-induced store Ca²⁺ release. Conversely, pretreatment with 50 μM clomiphene in Ca²⁺-free medium abolished the [Ca²⁺]_i increase induced by brefeldin A, thapsigargin or carbonylcyanide m-chlorophenylhydrazone. The 50 μM clomiphene-induced Ca²⁺release was unaltered by inhibiting phospholipase C with 2 μM 1-(6-((17β-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione (U73122). Trypan blue exclusion assay suggested that incubation with clomiphene (50 μM) for 2-15 min induced time-dependent decrease in cell viability by 10-50%. Collectively, the results suggest that clomiphene induced [Ca²⁺]_i increases in PC3 cells by releasing store Ca²⁺ from multiple stores in an phospholipase C-independent manner, and by activating Ca²⁺ influx; and clomiphene was of mild cytotoxicity.     

Photosensitizing action of isomeric zinc N-methylpyridylporphyrins in human carcinoma cells

The success of photodynamic therapy (PDT), as a minimally invasive approach, in treating both neoplastic and non-neoplastic diseases has stimulated the search for new compounds with potential application in PDT. We have previously reported that Zn(II) N-alkylpyridylporphyrins (ZnTM-2(3,4)-PyP⁴⁺ and ZnTE-2-PyP⁴⁺) can act as photosensitizers and kill antibiotic-resistant bacteria. This study investigated the photosensitizing effects of the isomers of ZnTMPyP⁴⁺ (ZnTM-2(3,4)-PyP⁴⁺) and respective ligands on a human colon adenocarcinoma cell line. At 10 μM and 30 min of illumination the isomeric porphyrins completely inhibited cell growth, and at 20 μM killed approximately 50% of the cancer cells. All these effects were entirely light-dependent. The isomers of the ZnTMPyP⁴⁺ and the respective ligands show high photosensitizing efficiency and no toxicity in the dark. Their efficacy as photosensitizers is comparable to that of hematoporphyrin derivative (HpD).     

Colocalization of NO and VIP in neurons of the submucous plexus in the rat intestine

Since very few previous studies have carried out the quantitative analysis for the colocalization of nitric oxide (NO) and vasoactive intestinal peptide (VIP) in the submucous neurons in the rat digestive tract, we applied in vivo treatment of colchicine to enhance the immunoreactivity and examined the colocalization of NO synthase (nNOS) and VIP in neurons of the submucous plexus throughout the rat digestive tract. The density of nNOS-containing neurons in the submucous plexus in the stomach corpus (103±25 cells/cm², n=3) and that in the antrum (157±9 cells/cm², n=3) were significantly lower than those in small and large intestine. However no difference was detected in the cell density among duodenum (1967±188 cells/cm², n=3), jejunum (2640±140 cells/cm², n=3), ileum (2070±42 cells/cm², n=3), proximal colon (2243±138 cells/cm², n=3) and distal colon (2633±376 cells/cm², n=3). The proportion of nNOS-immunoreactive (IR), nNOS/VIP-IR and VIP-IR neurons to the total number of submucous neurons was examined. nNOS/VIP-IR neurons comprised 45–55% of total number of submucous neurons from the duodenum to the proximal colon, however those comprised 66.4±5.1% in the distal colon. The results showed that the dense distribution of nNOS-containing neurons was found in the submucous plexus throughout the small and large intestine, and large population of submucous neurons co-stored nNOS and VIP.     

Mutagenicity of 8-methoxypsoralen and long-wave ultraviolet irradiation in V-79 Chinese hamster cells : A first approach to a risk estimate in photochemotheraphy

The effect of 8-methoxypsoralen (8-MOP) and long-wave ultraviolet irradiation (UVA) on cell killing and mutation induction was studied in V-79 Chinese hamster cells. No effect was observed after treatment with 8-MOP alone (50 μg/ml, 4 h), UVA alone (9000 J/m²), or 8-MOP metabolized by rat-livermicrosomes. Combined treatment with 8-MOP and UVA induced both cell killing and mutation. This was also observed under conditions approaching patient treatment with PUVA photochemotherapy with respect to the concentration of 8-MOP in the skin and the amount of UVA received by the epidermal cells. A simple relation proved to apply for mutation induction under different treatment conditions: 5.5 × 10⁻⁸ per J/m² per μg 8-MOP/ml. On this basis the mutation induction in dividing cells per session of PUVA-photochemotherapy amounts to 12.4 × 10⁻⁵, which is probably an over-estimation.     

Detection of heavy metal toxicity using cardiac cell-based biosensor

Biosensors incorporating mammalian cells have a distinct advantage of responding in a manner which offers insight into the physiological effect of an analyte. To investigate the potential applications of cell-based biosensors on heavy metal toxicity detection, a novel biosensor for monitoring electrophysiological activity was developed by light-addressable potentiometric sensor (LAPS). Extracellular field potentials of spontaneously beating cardiomyocytes could be recorded by LAPS in the range of 20 μV to nearly 40 μV with frequency of 0.5–3 Hz. After exposed to different heavy metal ions (Hg²⁺, Pb²⁺, Cd²⁺, Fe³⁺, Cu²⁺, Zn²⁺; in concentration of 10 μM), cardiomyocytes demonstrated characteristic changes in terms of beating frequency, amplitude and duration under the different toxic effects of ions in less than 15 min. This study suggests that, with the physiological monitoring, it is possible to use the cardiac cell-based biosensor to study acute and eventually chronic toxicities induced by heavy metal ions in a long-term and no-invasive way.     

Modulation of ERK 1/2 and p38 MAPK signaling pathways by ATP in osteoblasts: involvement of mechanical stress-activated calcium influx, PKC and Src activation

There is evidence that extracellular nucleotides, acting through multiple P2 receptors, may play an important role in the regulation of bone metabolism by activating intracellular signaling cascades. We have studied the modulation of mitogen-activated protein kinase (MAPK) signaling pathways and its relationship to changes in intracellular calcium concentration ([Ca²⁺]_i) induced by ATP in ROS-A 17/2.8 osteoblastic cells. ATP and UTP (10 μM) increased [Ca²⁺]_i by cation release from intracellular stores. We have found that when the cells are subsequently subjected to mechanical stress (medium perturbation), a transient calcium influx occurs. This mechanical stress-activated calcium influx (MSACI) was not observed after ADP stimulation, indicating that P2Y₂ receptor activation is required for MSACI. In addition, ERK 1/2 and p38 MAPK were activated by ATP in a dose- and time-dependent manner. This activation was almost completely blocked using neomycin (2.5 mM), an inhibitor of phosphoinositide-phospholipase C (PI-PLC), Ro 318220 (1 μM), a protein kinase C (PKC) inhibitor, and PP1 (50 μM), a potent and selective inhibitor of the Src-family tyrosine kinases. Ca²⁺-free extracellular medium (containing 0.5 mM EGTA) and the use of gadolinium (5 μM), which suppressed MSACI, prevented ERK 1/2 and p38 phosphorylation by ATP. Altogether, these results represent the first evidence to date suggesting that P2Y₂ receptor stimulation by ATP in osteoblasts sensitizes mechanical stress activated calcium channels leading to calcium influx and a fast activation of the ERK 1/2 and p38 MAPK pathways. This effect also involves upstream mediators such as PI-PLC, PKC and Src family kinases.     

Stimulation of platelet serotonin and Rb uptake by N-ethylmaleimide

The effects of N-ethylmaleimide (NEM) on mouse platelet serotonin (5-HT) and ⁸⁶Rb⁺ uptake were studied. The 5-HT transport system showed a biphasic response to increasing concentrations of NEM, with low concentrations (25–50 μM) stimulating and high concentrations (200–400 μM) inhibiting 5-HT transport. Fluoxetine, an inhibitor of the platelet 5-HT transporter, blocked NEM-induced stimulation of 5-HT transport. The kinetics of 5-HT uptake indicated that NEM (50 μM) markedly increased the maximal rate of 5-HT transport (V_max control = 28.4±1.4 pmol/10⁸ platelets/4 min vs V_max NEM = 64.5±9.5 pmol/10⁸ platelets/4 min but had no significant effect on the K_m value. Platelet Na⁺ K⁺ ATPase activity was determined by measuring ⁸⁶Rb⁺ uptake. Platelet ⁸⁶Rb⁺ uptake showed a biphasic response to NEM, with low concentrations (25–100 μM) significantly stimulating and high concentrations (400 μM) inhibiting uptake. These changes in platelet ⁸⁶Rb⁺ uptake paralleled the biphasic changes in 5-HT transport. In the presence of fluoxetine, 5-HT transport was markedly inhibited but no change in the ability of NEM to stimulate ⁸⁶Rb⁺ uptake was observed. These data suggest that low concentrations of NEM activate plasma membrane Na⁺ K⁺ ATPase which results in a marked stimulation of platelet 5-HT transport.     

Multilayer assembly of positively charged polyelectrolyte and negatively charged glucose oxidase on a 3D Nafion network for detecting glucose

In this paper, a novel amperometric glucose biosensor was constructed by alternative self-assembly of positively charged poly(diallydimethylammonium chloride) (PDDA) and negatively charged glucose oxidase (GOx) onto a 3D Nafion network via electrostatic adsorption. The amount of Nafion in the electrode and the number of the (PDDA/GOx)_n multilayers were optimized to develop a sensitive and selective glucose biosensor. Under optimal conditions, the glucose biosensor with (PDDA/GOx)₅ multilayers exhibited remarkable electrocatalytic activity, capable of detecting glucose with enhanced sensitivity of 9.55 μA/mM cm² and a commendably low detection limit of 20 μM (S/N = 3). A linear response range of 0.05–7 mM (a linear correlation coefficient of 0.9984, n = 20) was achieved. In addition, the glucose biosensor demonstrated superior selectivity towards glucose over some interferents, such as ascorbic acid (AA) and uric acid (UA), at an optimized detection potential of 0.6 V versus Ag/AgCl reference.     

Prostanoid formation in primary astroglial cell cultures: Ca-dependency and stimulation by A 23187, melittin and phospholipases A2 and C

Prostaglandin (PG) and thromboxane B₂ (TXB₂) biosynthesis was studied in cultured astrocytes from neonatal rat brain hemispheres. After two weeks of cultivation, prostanoids were formed with the spectrum: PGD₂ > TXB₂ > PGF₂ > PGE₂, as measured by specific radioimmunoassays. Under basal conditions PGD₂ biosynthesis (9.55 ng/mg protein/15 min) was in the same order of magnitude as the sum of the other prostanoids. The formation of prostanoids was stimulated in a concentration dependent manner (up to 6–10 fold) by the calcium ionophore A 23187 (0.01–10 μM) as well as by melittin (0.01–5 μg/ml), phospholipase A₂ (10–40 U/ml) and phospholipase C (0.01–1 U/ml). Basal and evoked PG and TXB₂ biosynthesis depended on the availability of Ca²⁺, as demonstrated in Ca²⁺ free incubation medium containing Na₂EDTA (1 μM), or with verapamil (100 μM) and 3,4,5-trimethoxybenzoic acid-8-(diethylamino)-octylester-HCl (TMB-8, 1–100 μM). Indomethacin (10 μM), mepacrine (100 μM) and p-bromophenacylbromide (50 μ M) inhibited basal and evoked PG formation. Thin-layer chromatography (TLC) detection after incubation of the cells with [³H]arachidonic acid (1 μCi/ml, for 60 min) confirmed the results obtained by radioimmunoassay. Incubation of [³H]arachidonic acid labelled cells with inonophore or phospholipases, followed by lipid extraction and TLC, showed that A 23187 liberated [³H]arachidonic acid predominantly from phosphatidylethanolamine, whereas phospholipase A₂ and C reduced mainly the labelling of the phosphatidyl-inositol/-choline fraction. Potassium depolarization of the cells did not enhance prostanoid formation. Similarly, drugs with affinity to - or β-adrenoceptors, or to dopamine-, 5-hydroxytryptamine-, muscarine-, histamine-, glutamate-, aspartate-, GABA, adenosine- and opioid-receptors failed to stimulate prostanoid biosynthesis. Also compounds like angiotensin, bradykinin and thrombin were ineffective in this respect.

In conclusion, our results confirm that cultured astrocytes possess the complete pattern of enzymes necessary for prostanoid formation and hence might play a crucial role in brain prostanoid biosynthesis. Stimulation of prostanoid biosynthesis involves Ca²⁺-dependent activation of phospholipase A₂, cyclooxygenase reaction and further PG metabolism. However, the endogenous stimulus for enhanced prostanoid synthesis in the brain still has to be established.     

The use of different eye regions in the mantis shrimp Hemisquilla californiensis Stephenson, 1967 (Crustacea: Stomatopoda) for detecting objects

A behavioral assay was used to assess the ability of the stomatopod Hemisquilla californiensis to perceive and respond to a moving target under different wavelengths and intensities of light illumination. Subjects responded to targets rotating horizontally across their visual field by a brief startle response of their eyes or antennules but did not track the targets. Under white light responses were elicited down to a light intensity of 0.9 μW cm^− 2. Responses were seen in blue light at intensities as low as 0.5 μW cm^− 2, and in green light down to 1.0 μW cm^− 2. The animals were less sensitive to red light, with no responses seen at intensities below 3.0 μW cm^− 2. Subjects did not respond to the targets at all under infrared light. This response pattern mirrors the computed sensitivity spectrum of ommatidia in the species' peripheral hemispheres but not that in most of the central bands. We conclude that this species uses the monochromatic vision in the peripheral hemispheres of its eyes to recognize objects and that the sharply tuned color receptors of the central band serve to add supplemental information if light conditions allow.     

Enhancement of growth and gymnodimine production by the marine dinoflagellate, Karenia selliformis

Because of its novel bioactive properties the production of gymnodimine for use as a pharmaceutical precursor has aroused interest. The dinoflagellate, Karenia selliformis produces gymnodimine when grown in bulk culture using GP + selenium medium but the growth rates (μ) and levels of gymnodimine are low (μ, 0.05 days⁻¹; gymnodimine 250 μg L⁻¹ max). We describe the effects of organic acid additions (acetate, glycolate, alanine and glutamate additions and combinations of these) in enhancing growth and gymnodimine production in axenic cultures. The most effective organic acid combinations in decreasing order were: glycolate/alanine > acetate > glycolate. Glycolate/alanine optimised gymnodimine production by prolonging growth (maximum cell yield, 1.76 × 10⁵ cells mL⁻¹; gymnodimine, 1260 μg L⁻¹; growth rate (μ), 0.2 days⁻¹) compared to the control (growth maximum cell yield, 7.8 × 10⁴ cells mL⁻¹; gymnodimine, 780 μg L⁻¹; μ, 0.17 days⁻¹). Acetate enhanced gymnodimine by stimulating growth rate (μ, 0.23 days⁻¹) and the large concentration of gymnodimine per cell (16 pg cell⁻¹ cf. 9.8 pg cell⁻¹ for the control) suggests a role for this compound in gymnodimine biosynthesis. Amending culture media with Mn²⁺ additions resulted in slightly decreased growth in control cultures and increased the gymnodimine while in glycolate/alanine cultures growth was stimulated but gymnodimine production decreased. The results suggest that the organic acid can enhance gymnodimine production by either enhancing growth maximum or the biosynthetic pathway.     

Crystal and microparticle effects on MDCK cell superoxide production: oxalate-specific mitochondrial membrane potential changes

We have previously shown that crystals of calcium oxalate (COM) elicit a superoxide (O₂⁻) response from mitochondria. We have now investigated: (i) if other microparticles can elicit the same response, (ii) if processing of crystals is involved, and (iii) at what level of mitochondrial function oxalate acts. O₂⁻ was measured in digitonin-permeabilized MDCK cells by lucigenin (10 μM) chemiluminescence. [¹⁴C]-COM dissociation was examined with or without EDTA and employing alternative chelators. Whereas mitochondrial O₂⁻ in COM-treated cells was three- to fourfold enhanced compared to controls, other particulates (uric acid, zymosan, and latex beads) either did not increase O₂⁻ or were much less effective (hydroxyapatite +50%, p < 0.01), with all at 28 μg/cm². Free oxalate (750 μM), at the level released from COM with EDTA (1 mM), increased O₂⁻ (+50%, p < 0.01). Omitting EDTA abrogated this signal, which was restored completely by EGTA and partially by ascorbate, but not by desferrioxamine or citrate. Omission of phosphate abrogated O₂⁻, implicating phosphate-dependent mitochondrial dicarboxylate transport. COM caused a time-related increase in the mitochondrial membrane potential (Δψ_m) measured using TMRM fluorescence and confocal microscopy. Application of COM to Fura 2-loaded cells induced rapid, large-amplitude cytosolic Ca²⁺ transients, which were inhibited by thapsigargin, indicating that COM induces release of Ca²⁺ from internal stores. Thus, COM-induced mitochondrial O₂⁻ requires the release of free oxalate and contributes to a synergistic response. Intracellular dissociation of COM and the mitochondrial dicarboxylate transporter are important in O₂⁻ production, which is probably regulated by Δψ_m.     

The non-specific inhibition of enzymes by environmental pollutants: a study of a model system towards the development of electrochemical biosensor arrays

Previous research has shown that lactate dehydrogenase (LDH) was competitively inhibited by pentachlorophenol (PCP) and a modified assay produced a detection limit of 1 μM (270 μg l⁻¹). This work used spectrophotometric rate-determination but in order to move towards biosensor development the selected detection method was electrochemical. The linkage of LDH to lactate oxidase (LOD) provided the electroactive species, hydrogen peroxide. This could be monitored using a screen-printed carbon electrode (SPCE) incorporating the mediator, cobalt phthalocyanine, at a potential of +300 mV (vs. Ag/AgCl). A linked LDH/LOD system was optimised with respect to inhibition by PCP. It was found that the SPCE support material, PVC, acted to reduce inhibition, possibly by combining with PCP. A cellulose acetate membrane removed this effect. Inhibition of the system was greatest at enzyme activities of 5 U ml⁻¹ LDH and 0.8 U ml⁻¹ LOD in reactions containing 246 μM pyruvate and 7.5 μM NADPH. PCP detection limits were an EC₁₀ of 800 nM (213 μg l⁻¹) and a minimum inhibition detectable (MID) limit of 650 nM (173 μg l⁻¹). The inclusion of a third enzyme, glucose dehydrogenase (GDH), provided cofactor recycling to enable low concentrations of NADPH to be incorporated within the assay. NADPH was reduced from 7.5 to 2 μM. PCP detection limits were obtained for an assay containing 5 U ml⁻¹ LDH, 0.8 U ml⁻¹ LOD and 0.1 U ml⁻¹ GDH with 246 μM pyruvate, 400 mM glucose and 2 μM NADPH. The EC₁₀ limit was 150 nM (39.9 μg l⁻¹) and the MID was 100 nM (26.6 μg l⁻¹). The design of the inhibition assays discussed has significance as a model for other enzymes and moves forward the possibility of an electrochemical biosensor array for pollution monitoring.     

Effect of melanin on netilmicin-induced inhibition of collagen biosynthesis in human skin fibroblasts

It is known that various drugs form complexes with melanins and that melanins are abundant constituents of the inner ear. In this study, we determined whether the aminoglycoside antibiotic, netilmicin, interacts with melanin and how this process affects collagen biosynthesis in cultured human skin fibroblasts. The obtained results indicate that netilmicin forms stable complexes with melanin characterized by the association constants K₁  10⁶ M⁻¹ and K₂  10³ M⁻¹. We have suggested that prolidase, an enzyme involved in collagen metabolism, may be one of the targets for aminoglycoside-induced inhibition of collagen biosynthesis. We found that netilmicin strongly induced inhibition of prolidase activity (IC₅₀ < 5 μM) and collagen biosynthesis (IC₅₀  10 μM). At 10 μM concentration of netilmicin, prolidase activity in human skin fibroblasts was inhibited by about 80% and DNA biosynthesis—only by about 25%. Melanin at 100 μg/mL produced about 30% inhibition of collagen biosynthesis and about 30% inhibition of prolidase activity in cultured fibroblasts. However, the addition of melanin (100 μg/mL) to netilmicin-treated cells (10 μM) restored the prolidase activity in fibroblasts to almost 100% of control values and partially reversed the inhibitory action of the drug on collagen and DNA biosynthesis. The data suggest that the ability of netilmicin to form stable complexes with melanin may prevent its toxicity on prolidase activity and collagen biosynthesis.     

A study in UF-membrane reactor on activity and stability of nitrile hydratase from Microbacterium imperiale CBS 498-74 resting cells for propionamide production

The bioconversion of propionitrile to propionamide was catalysed by nitrile hydratase (NHase) using resting cells of Microbacterium imperiale CBS 498-74 (formerly, Brevibacterium imperiale). This microorganism, cultivated in a shake flask, at 28 °C, presented a specific NHase activity of 34.4 U mg_DCW⁻¹ (dry cell weight). The kinetic parameters, K_m and V_max, tested in 50 mM sodium phosphate buffer, pH 7.0, in the propionitrile bioconversion was evaluated in batch reactor at 10 °C and resulted 21.6 mM and 11.04 μmol min⁻¹ mg_DCW⁻¹, respectively. The measured apparent activation energy, 25.54 kJ mol⁻¹, indicated a partial control by mass transport, more likely through the cell wall.

UF-membrane reactors were used for kinetic characterisation of the NHase catalysed reaction. The time dependence of enzyme deactivation on reaction temperature (from 5 to 25 °C), on substrate concentrations (from 100 to 800 mM), and on resting cell loading (from 1.5 to 200 μg  ml⁻¹) indicated: lower diffusional control (E_a=37.73 kJ mol⁻¹); and NHase irreversible damage caused by high substrate concentration. Finally, it is noteworthy that in an integral reactor continuously operating for 30 h, at 10 °C, 100% conversion of propionitrile (200 mM) was attained using 200 μg  ml⁻¹ of resting cells, with a maximum volumetric productivity of 0.5 g l⁻¹ h⁻¹.