Effects of Salmonella typhimurium infection and ofloxacin treatment on glucose and glutamine metabolism in Caco-2/TC-7 cells

The effects of both Salmonella typhimurium infection and 5 mM ofloxacin treatment on 2 mM glutamine and 5 mM glucose metabolism in the enterocyte-like Caco-2/TC-7 cell line were studied. These cells utilized glutamine (212.07 +/- 16.75 [mean +/- standard deviation] nmol per h per 10(6) viable cells) and, to a lesser extent, glucose (139.63 +/- 11.52 nmol per h per 10(6) viable cells). Metabolism of these substrates in Caco-2/TC-7 cells resembled that in rat, pig, or human enterocytes. Infection by S. typhimurium C53-enhanced glucose and glutamine substrate utilization by 32 and 22%, respectively and enhanced glucose and glutamine substrate oxidation by eight- and twofold, respectively. These increases in glucose and glutamine metabolism (especially glucose metabolism) were due in part to the metabolism of intracellular bacteria and/or to the activation of cellular metabolism. Substrate metabolism (especially glucose metabolism) in C53-infected cells was partially reduced by treatment with ofloxacin. It was concluded that cellular fuel metabolism is stimulated at the earliest stage of infection (3 to 4 h) and that treatment with 5 mM ofloxacin does not completely restore substrate metabolism to the levels observed in uninfected cells, possibly because this treatment does not eradicate intracellular S. typhimurium completely.     

Possible molecular mechanism of loss of homologous and heterologous gap junctional intercellular communication in rat liver epithelial cell lines

We have identified beta-galactosidase activity in purified bovine rod outer segments (ROS), using rho-nitrophenyl-beta-D-galactopyranoside (PNPG) and chlorophenol red-beta-D-galactopyranoside (CPRG) as substrates. This glycosylhydrolase activity did not appear to represent contamination from other retinal subcellular fractions, based upon the relative specific activities of beta-galactosidase vs. other hydrolases (N-acetyl-beta-glucosaminidase, alpha- and beta-mannosidase, alpha-fucosidase, and acid phosphatase) in bovine retina and ROS homogenates. Using PNPG as a substrate, two pH optima were observed (at 3.5 and 5.5), while the hydrolysis of CPRG exhibited a single, broad pH optimum centered at 5.5. In contrast, hydrolysis of PNPG and CPRG by retinal homogenates exhibited single pH optima, at 3.5 and 5.5., respectively. ROS beta-galactosidase activity increased linearly with time, temperature, and protein concentration, and obeyed Michaelis-Menten kinetics with both substrates. For PNPG, Vmax approximately 88 nmol/h/mg protein and the apparent Km approximately 147 microM. For CPRG, Vmax approximately 33 nmol/h/mg protein and the apparent Km approximately 50 microM. ROS beta-galactosidase activity was affected by carbohydrates and their derivatives: glucose, fucose, sucrose, maltose and N-acetyl-galactosamine were found to stimulate the activity, while D-galactono-gamma-lactone and, to a lesser extent, D-galactose were inhibitory. The enzyme activity also was slightly stimulated by [Cl-] and markedly by dithiothreitol (DTT), while rho-chloro-mercuribenzoic acid (PCMB) and rho-hydroxymercuribenzoic acid (PHMB) inactivated the enzyme. In addition, the enzymatic activity was also found to be differentially sensitive to various anionic and nonionic detergents. However, n-octyl-beta-D-glucoside was slightly stimulatory.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification and characterization of an alpha1,2,-L-fucosyltransferase, which modifies the cytosolic protein FP21,from the cytosol of Dictyostelium

A novel fucosyltransferase (cFTase) activity has been enriched over 10(6)-fold from the cytosolic compartment of Dictyostelium based on transfer of [3H]fucose from GDP-[3H]fucose to Galbeta1,3 GlcNAc beta-paranitrophenyl (paranitrophenyl-lacto-N-bioside or pNP-LNB). The activity behaved as a single component during purification over DEAE-, phenyl-, Reactive Blue-4-, GDP-adipate-, GDP-hexanolamine-, and Superdex gel filtration resins. The purified activity possessed an apparent Mr of 95 X 10(3), was Mg2+-dependent with a neutral pH optimum, and exhibited a Km for GDP-fucose of 0.34 microM, a Km for pNP-LNB of 0.6 mM, and a Vmax for pN-P-LNB of 620 nmol/min/mg protein. SDS-polyacrylamide gel electrophoresis analysis of the Superdex elution profile identified a polypeptide with an apparent Mr of 85 X 10(3), which coeluted with the cFTase activity and could be specifically photolabeled with the donor substrate inhibitor GDP-hexanolaminyl-azido-125I-salicylate. Based on substrate analogue studies, exoglycosidase digestions, and co-chromatography with fucosylated standards, the product of the reaction with pNP-LNB was Fucalpha1, 2Galbeta1,3GIcNAcbeta-pNP. The cFTase preferred substrates with a Galbeta1,3linkage, and thus its acceptor substrate specificity resembles the human Secretor-type alpha1,2- FTase. Afucosyl isoforms of the FP21 glycoprotein, GP21-I and GP21-II, were purified from the cytosol of a Dictyostelium mutant and found to be substrates for the cFTase, which exhibited an apparent K(m) of 0.21 microM and an apparent V(max) of 460 nmol/min/mg protein toward GP21-II. The highly purified cFTase was inhibited by the reaction products Fucalpha1,2Galbeta1,3GlcNAcbeta-pNP and FP21-II. FP21-I and recombinant FP21 were not inhibitory, suggesting that acceptor substrate specificity is based primarily on carbohydrate recognition. A cytosolic location for this step of FP21 glycosylation is implied by the isolation of the cFTase from the cytosolic fraction, its high affinity for its substrates, and its failure to be detected in crude membrane preparations.     

Preparation and transplantation of photoreceptor sheets

PURPOSE: Photoreceptor (PR) transplantation may be a treatment for blindness secondary to PR degeneration. We studied different technical aspects of PR-sheet preparation. METHODS: Geographic variation in the thickness of the cat PR layer (from the outer segments to the outer plexiform layer) and inner retina (width of the remainder of the retina) was studied. PR sheets (cat and human) were prepared through gelatin embedding and subsequent vibratoming or excimer laser ablation. Cat PR sheets were evaluated after transplantation. RESULTS: The thickness of the cat PR layer and inner retina varied in different regions. The superior central retina, including the area centralis, was thickest (PR layer: 115-123 microm, entire retina: 225-230 microm, in fixed tissue). The peripheral retina was approximately 40% thinner than the center. Fresh retina was approximately 7.9% thicker than the fixed retina. Both vibratomy and excimer laser ablation removed the inner retina, leaving a PR-layer sheet with good morphology. To produce good quality PR sheets with vibratomy, use of different gelatin concentrations (2% to 35%) at various stages of sheet preparation was crucial. To produce PR sheets of uniform thickness with excimer laser ablation, control of fluid on the retinal surface was critical. Twenty-four hours after PR transplantation surgery, donor PR cells were well oriented and in close contact with host retinal pigment epithelial cells. Gelatin supporting the transplant dissolved as early as 100 min after surgery. CONCLUSIONS: We confirmed and expanded the work of previous investigators and showed that cat and human PR sheets can be manufactured using vibratomy or excimer laser ablation. This preparation provides a well oriented and organized PR cell layer after transplantation.     

TECHNICAL NOTE: measurement of cAMP-dependent protein kinase activity using a fluorescent-labeled kemptide

BACKGROUND: Traditional protein kinase assays include the use of [32P] labeled ATP as phosphate donor and a substrate protein or peptide as phosphoreceptor. Since this approach has a number of drawbacks in addition to generating ionizing radiation, several non-isotopic methods have been developed. Although shown to reflect the activity of purified enzymes, none have been demonstrated to detect physiological changes in endogenous enzyme activity in cell homogenates. METHODS: Studies were performed to examine the kinetics, reproducibility, and optimal assay conditions of a novel non-radioisotopic kinase assay that detects PKA activity by phosphorylation of the peptide substrate Kemptide covalently bound to a fluorescent molecule (f-Kemptide). Basal and agonist-induced PKA activity in epithelial cell homogenates was measured. RESULTS: The kinetics of f-Kemptide were similar to the standard radioisotopic method with intraassay and interassay variations of 5.6 +/- 0.8% and 14.3 +/- 2.6%, respectively. Neither fluorescence quenching nor enhancing effects were found with consistent amounts of homogenate protein. Specific PKA activity was determined as the IP20-inhibitable fraction to account for nonspecific phosphorylation, perhaps due to S6 kinase or a similar enzyme. The basal activity of 38% of total PKA in A6 cells increased by 84% after exposure to vasopressin and by 58% after short exposure to forskolin. In T84 cells exposed to VIP there was a 360% increase over basal activity. CONCLUSIONS: These results show that f-Kemptide exhibits acceptable kinetics, and that the assay system can quantitatively and reproducibly measure basal and stimulated PKA activity in cell homogenates.     

Biotransformation of estradiol in the human keratinocyte cell line HaCaT: metabolism kinetics and the inhibitory effect of ethanol

PURPOSE: The aim of our study was to investigate the kinetics of beta-estradiol (E2) metabolism in the human keratinocyte cell line HaCaT and to estimate the effect of the potential inhibitor ethanol on the biotransformation reaction. METHODS: The formation rates of estrone (E1) in dependence on substrate concentrations were determined in HaCaT cells using tritium labelled E2. Experiments were conducted with and without addition of dehydroepiandrosterone (DHEA) and ethanol. Possible toxic effects on the cells due to ethanol were investigated by cytotoxicity tests. RESULTS: The metabolism of E2 in HaCaT cells exhibited Michaelis-Menten kinetics with Km and Vmax values of 3.5 microM and 216 pmol x mg(-1) protein x h(-1), respectively. The reaction was inhibited by DHEA and ethanol. The alcohol showed a reversible competitive inhibition mechanism for concentrations of 4 to 8% (v/v). Lower ethanol concentrations had no effect, whereas levels > or =10% significantly decreased cell viability leading to a different inhibition mechanism. CONCLUSIONS: The HaCaT cell line seems to be a suitable model for studying enzyme kinetics equivalent to the human skin. The concentration dependent inhibitory effect of ethanol observed in this cell line may be relevant for the transdermal E2 application in patients.     

Indocyanine green: intracellular uptake and phototherapeutic effects in vitro

Indocyanine green (ICG; absorption peak in human plasma 805 nm) was investigated for ICG-mediated phototherapy in vitro. The cellular uptake of ICG (1 microM-50 microM) into HaCaT keratinocytes after an incubation period of 24 h increased up to an intracellular ICG concentration of 12.1 +/- 1.3 nmol per 10(6) cells. To examine dose dependent phototoxic effects in vitro, keratinocytes were incubated with 0 microM-50 microM ICG for 24 h and irradiated by a diode laser (805 nm) with different energy densities (0, 12, 24, 48 J cm-2). All applied ICG concentrations except for 5 microM yielded a cell killing effect in combination with irradiation depending significantly on ICG concentration and light dose. Cell viability for dark control and cells incubated with 50 microM ICG and irradiated with 48 J cm-2 was 0.82 +/- 0.15 and 0.07 +/- 0.02, respectively. Sodium azide (100 mM), a quencher of reactive oxygen species, inhibited significantly the cell killing using 50 microM ICG and 24 J cm-2. Taken together, photoactivation of ICG by irradiation with a diode laser was shown to induce effectively cell killing of HaCaT keratinocytes. Moreover, this effect was inhibited by sodium azide, thus irradiation of ICG might induce a photodynamic reaction.     

Structure-activity relationships of cysteine esters and their effects on thiol levels in rat lung in vitro

Pretreatment with cysteine esters increases cysteine (CySH) levels in rat lung and protects against the lethal effects of inhaled perfluoroisobutene in vivo. There are marked differences in the duration of protection achieved with different cysteine esters. In this study we have compared the uptake and metabolism of CySH, N-acetyl cysteine (NAc), cysteine esters and cystine esters in vitro using rat lung and liver homogenates and lung slices. Liver homogenates metabolized CySH and cysteine esters faster than lung homogenates. The half life (T1/2) of CySH in lung was 58.8 +/- 17.3 min and in liver was 14.0 +/- 1.6 min (mean +/- SEM). T1/2 of the esters in lung ranged between 6.5 and 12.1 min and in liver between 1.9 and 5.3 min. Cysteine tertiary butyl ester, which does not protect in vivo, was not hydrolysed to CySH by lung or liver homogenates. All esters increased and prolonged intracellular CySH concentrations in lung slices to a much greater extent than CySH itself. NAc did not raise intracellular CySH above that of the controls and no NAc appeared within the slice. After CySH incubation intracellular CySH was 0.9 +/- 0.1 nmol/mg wet wt at 10 min whereas after incubation with the esters it ranged between 2.60 and 3.65 nmol/mg wet wt. Cysteine cyclohexyl ester prolonged the increase of CySH the longest and cysteine methyl ester the shortest. CySH levels with cysteine cyclohexyl ester were 2.74 +/- 0.15 and 4.13 +/- 0.37 nmol/mg wet wt at 10 and 60 min, respectively, whereas with cysteine methyl ester, CySH levels were 2.60 +/- 0.5 and 1.25 +/- 0.08 nmol/mg wet wt at similar times. Cystine esters increased intracellular concentrations of both cystine and CySH. CySH concentrations ranged between 2.92 and 3.19 nmol/mg wet wt and cystine between 1.39 and 1.47 nmol/mg wet wt at 60 min. The elevation and duration of CySH in lung slices is well correlated with the duration of protection against perfluoroisobutene achieved in vivo.     

Brain bilirubin content is increased in P-glycoprotein-deficient transgenic null mutant mice

P-glycoprotein (P-gp), encoded by the mdr1a gene, is an ATP-dependent plasma membrane protein that is expressed in abundance on the blood-brain barrier (BBB). P-gp limits the CNS influx and retention of a variety of lipophilic compounds. We hypothesized that brain bilirubin content after an i.v. bilirubin infusion would be increased in P-gp-deficient mdr1a null mutant transgenic mice (mdr1a(-/-)) compared with controls. Eighteen mdr1a(-/-) null mutant and 18 P-gp-sufficient wild type mice (+/+) were anesthetized and 50 mg/kg bilirubin infused through the tail vein. Brain bilirubin content (mean +/- SEM) 10 min after infusion was significantly higher in mdr1a(-/-) (18.1 +/- 2.4 nmol/g) compared with (+/+) mice (10.4 +/- 1.0 nmol/g). Brain bilirubin content declined 60 min after infusion but remained higher in mdr1a(-/-) (10.3 +/- 1.4 nmol/g) compared with (+/+) mice (5.3 +/- 0.9 nmol/g). Brain bilirubin clearance did not differ between groups (t 1/2 approximately 55 min). We conclude that P-gp-deficient mdr1a(-/-) mice have significantly higher brain bilirubin content compared with controls after an i.v. bilirubin load. These data suggest that 1) bilirubin is a substrate for P-gp and 2) the increased brain bilirubin content in mdr1a(-/-) mice is due to enhanced brain bilirubin influx. We speculate that BBB P-gp provides a protective effect against bilirubin neurotoxicity by reducing brain bilirubin influx.     

The activity of MAO A and B in rat renal cells and tubules

The present study reports on the presence of type A and B monoamine oxidase (MAO) activity and their sensitivity to selective MAO-A and MAO-B inhibition by Ro 41-1049 and lazabemide, respectively, in homogenates of isolated rat renal tubules. Non-linear analysis of the saturation curve of H-5-hydroxytryptamine (3H-5-HT ) deamination revealed a Km of 351+/-71 microM (n=4) and a Vmax of 25+/-2 nmol mg protein(-1) h(-1). Deamination of 14C-beta-phenylethylamine (14C-beta-PEA) was also a saturable process yielding Km values of 58+/-12 microM and Vmax values of 24+/-2 nmol mg protein(-1) h(-1). Ro 41-1049 produced a concentration-dependent inhibition of 3H-5-HT deamination with a Ki of 24 nM. Deamination of 14C-beta-PEA was found to be reduced by lazabemide in a concentration-dependent manner with a Ki value of 17 nM. The effect of these selective MAO inhibitors on dopamine fate and DOPAC formation in isolated tubular epithelial cells was also studied. In these studies a clear inhibition of DOPAC formation was observed with Ro 41-1049 (250 nM), while 250 nM lazabemide was found not to increase the accumulation of newly-formed DA in those tubular epithelial cells loaded with 50 microM L-DOPA. In conclusion, the results presented here confirm the presence of both MAO-A and MAO-B activity in renal tubular epithelial cells, that MAO-A is the predominant enzyme involved in the deamination of the natriuretic hormone dopamine and that the deamination of newly-formed dopamine is a time-dependent process which occurs early after the decarboxylation of L-DOPA.     

Oxidation of acetate and octanoate and its relation to glucose metabolism in contracting porcine carotid artery

The oxidation of octanoate and acetate was measured in segments of porcine carotid arteries to ascertain whether the oxidation of exogenous fatty acid substrates (acetate and octanoate) is augmented during contraction induced by K(+)-depolarization. The oxidation of acetate increased from 7 +/- 1 to 14 +/- 2 nmol/min/g (P < 0.01) during sustained isometric contraction. Octanoate oxidation increased from 11 +/- 1 to 14 +/- 1 nmol/min/g (P < 0.05). The rate of oxidation of neither acetate nor octanoate was affected by the presence or absence of glucose either in resting or contracting arteries Acetate or octanoate oxidation could account for the majority of O2 consumption during contraction. Octanoate but not acetate inhibited glucose uptake and glycolysis in resting muscles. In contrast to augmented acetate and octanoate metabolism during contraction, there was a "down-regulation" of glucose metabolism in contracting muscles as evidenced by a decrease in the rate of glucose uptake, glycolysis and lactic acid production during sustained isometric contraction. Thus, contractile activation of vascular smooth muscle is associated with a shifting pattern of substrate utilization. Exogenous acetate or octanoate can serve as the primary oxidative substrate during sustained isometric contraction.     

Retinal pigment epithelium cell culture on thin biodegradable poly(DL-lactic-co-glycolic acid) films

Thin films of 50:50 and 75:25 poly(DL-lactic-co-glycolic acid) (PLGA) were manufactured with a controlled thickness of less than 10 microm. The effect of PLGA copolymer ratio on in vitro cell attachment, proliferation, morphology, and tight junction formation was evaluated using a human D407 retinal pigment epithelium (RPE) cell line. Almost complete cell attachment was achieved on both PLGA films after 8 h of cell seeding, which was comparable to that on tissue culture polystyrene (TCPS) controls. The initial cell seeding density affected attachment, and the optimal value for 50:50 PLGA was 25000 cells cm(-2). After 7 days of in vitro culture, cell density on 50:50 and 75:25 PLGA films increased 45 and 40 folds, respectively, and a 34-fold increase was observed on TCPS. The RPE cells cultured on PLGA films at confluence had a characteristic cobblestone morphology. Confluent RPE cells also developed normal tight junctions in vitro which were concentrated mainly at the apical surfaces of cell-cell junctions. These results demonstrated that thin biodegradable PLGA films can provide suitable substrates for human RPE cell culture, and may serve as temporary carriers for subretinal implantation of organized sheets of RPE.     

Cultivation of Escherichia coli with mixtures of 3-phenylpropionic acid and glucose: steady-state growth kinetics

The fate of pollutants in the environment is affected by the presence of easily degradable carbon sources. As a step towards understanding these complex interactions, a model system was explored: the degradation of mixtures of glucose (i.e., an easily degradable substrate) and 3-phenylpropionic acid (3ppa) (a model pollutant) by Escherichia coli ML 30 was studied systematically in carbon-limited continuous culture. The two substrates were always consumed simultaneously regardless of the dilution rate applied. Even at dilution rates higher than the maximum specific growth rate for 3ppa (0.35 +/- 0.05 h-1), the two carbon substrates were utilized together. When cells were grown at a constant dilution rate with different mixtures of 3ppa and glucose, in which 3ppa contributed between 5 and 90% of carbon substrate in the feed medium, the steady-state concentrations of 3ppa and glucose were approximately proportional to the ratio of the two substrates in the feed medium. When cells were cultivated at different dilution rates with a 1:1 mixture (based on carbon) of glucose and 3ppa, an overall maximum specific growth rate of 0.90 +/- 0.05 h-1 and a Monod substrate saturation constant for 3ppa (Ks) of 600 to 700 micrograms liter-1, similar to that measured during growth with 3ppa alone, fitted the experimentally determined steady-state 3ppa concentrations. However, due to the highly differing substrate affinity constants for 3ppa and glucose (Ks approximately 30 to 70 micrograms liter-1), the total steady-state carbon concentration in the culture at a constant dilution rate was determined mainly by the steady-state 3ppa carbon concentration, and it increased with increasing proportions of 3ppa in the feed medium.     

Kinetic studies of Bacillus polymyxa nitrogenase

Nitrogenase from the facultative anaerobe Bacillus polymxa was separated into its component proteins, which were recombined in the ratio that produced optimal specific activity (125 to 175 nmol of C2H2 reduced/min per mg of total protein). The apparent Michaelis constants (Km)for the magnesium adenosine triphosphate complex, reducible substrates azide, acetylene, and N2 and the nonphysiological electron donor hydrosulfite (S2O42-) were determined to be 0.7, 0.7, 0.2, 0.06, and 0.03 MM, respectively. These apparent Km values are in reasonable agreement with those reported for the nitrogenases of Azotobacter vinelandii and Klebsiella pneumoniae. Either a total lack of cooperativity between binding sites or a single binding site for reducible substrates is indicated by analysis of Hill plots. Hill plot slopes of approximately 1.7 suggest that multiple binding sites exist for both ATP and S2O42-.     

Induction of subcutaneous tissue fibrosis in newborn mice by transforming growth factor beta--simultaneous application with basic fibroblast growth factor causes persistent fibrosis

OBJECTIVE: To determine the effect of serum P on endometrial histology in stimulated cycles. DESIGN: Prospective clinical study. SETTING: Community hospital-based donor oocyte program. PATIENT(S): Fertile young oocyte donors and infertile donor oocyte recipients. INTERVENTION(S): Oocyte donors underwent gonadotropin stimulation after midluteal pituitary suppression. Endometrial biopsies were obtained at the time of oocyte retrieval. MAIN OUTCOME MEASURE(S): Endometrial histology and serum P levels in oocyte donors. Pregnancy and implantation rates in oocyte recipients. RESULT(S): Thirteen biopsy specimens (52.0%) showed in-phase mixed proliferative pattern (days 14 to 15), whereas 12 (48.0%) were secretory (days 16 to 17). On the day of hCG, subjects with secretory endometrium had higher P of 1.7 ng/mL (5.4 nmol/L) than women with the mixed pattern (0.8 ng/mL [2.5 nmol/L]). Progesterone > or = 0.9 ng/mL had a 78.6% positive predictive value for secretory transformation. In 75.0% of cycles with secretory endometrium, P was > or = 0.9 ng/mL, (2.9 nmol/L) as early as 2 days before hCG. Both mixed and secretory patterns were associated with similar clinical pregnancy rates (57.1% and 60.0%, respectively) and delivery rates (38.1% and 50.0%, respectively) in recipients. CONCLUSION(S): Subtle elevation of P induced secretory endometrial transformation without reduction in embryo viability.     

Rat liver catechol-O-methyltransferase kinetics and assay methodology

In mammals, catechol-O-methyltransferase (COMT) is distributed throughout various organs, the highest activities being found in the liver and kidney. However, comparisons of the kinetic parameters are difficult to perform, since the experimental procedures in the enzyme assay vary quite considerably. The present work was aimed at studying the optimal liver COMT assay conditions for determining the kinetics of the enzyme. The COMT assay was performed with liver homogenates from 60 days old male Wistar rats with adrenaline (AD) as the substrate. Time course experiments using 100 microM S-adenosyl-L-methionine (SAMe) and 300 microM AD showed linearity of O-methylation reaction upto 10 min. Using 100 microM SAMe, Vmax (nmol mg protein-1 h-1) and Km (microM) values progressively decreased respectively from 22.1 and 104.8 at 5 min down to 5.8 and 24.62 at 60 min incubation periods. This decrease was not due to end-product inhibition. Using 2500 microM AD, Km values (microM) for the methyl donor SAMe increased progressively from 174 at 5 min upto 1192.5 at 60 min; upto 30 min of incubation Vmax values did not change. When a 5 min incubation period and 500 microM SAMe were used, Vmax and Km values for liver COMT were 63.4 nmol mg protein-1 h-1 and 261.1 microM, respectively. It is concluded that an incubation period of 5 min and a SAMe concentration of 500 microM provide optimal conditions for the liver homogenate COMT assay.     

On epidemiology and geographic information systems: a review and discussion of future directions

Leu-enkephalin radiolabelled at the N-terminal tyrosine by two different methods was presented to isolated perfused rat livers. Approximately 10% of a pulse of tritiated Leu-enkephalin was taken up first-pass; this was increased to 62% when the peptide was iodinated with Bolton and Hunter reagent. Uptake of both forms of radiolabelled Leu-enkephalin was inhibited by taurocholate in a concentration-dependent manner. The proportion of internalised radioactivity secreted into bile also differed but in both cases showed a very rapid time-course similar to that of [24-(14)C]taurocholate and suggestive of non-endocytic transfer via membrane transport proteins. Pre-perfusion with the aminopeptidase inhibitor bestatin increased uptake of 3H-labelled Leu-enkephalin from 10% to 23%; no further increase occurred when the endopeptidase 24.11 inhibitor thiorphan was also present. On infusion of the native peptide into rat livers, 80% of Leu-enkephalin immunoreactivity was lost between the pre- and post-hepatic perfusate; this was reduced to 65% in the presence of 10(-5) M bestatin. The almost total release of the N-terminal tyrosine from 3H-labelled Leu-enkephalin which escaped first-pass uptake confirmed that substantial sinusoidal metabolism had occurred. Low levels of aminopeptidase N were visualised in the sinusoidal membrane using a specific monoclonal antibody coupled to peroxidase staining. Thus, hepatic inactivation of Leu-enkephalin is primarily via hydrolysis mediated by cell surface peptidase (including aminopeptidases) whilst uptake of the intact peptide, probably by a bile salt transport protein, is quantitatively minor unless the N-terminus is blocked by Bolton and Hunter reagent or peptidase inhibitors are present.     

Photosensitization of skin-derived cell lines by Dimegin [2,4-di-(alpha-methoxyethyl)-deuteroporphyrin IX] in vitro

The deuteroporphyrin-IX derivative Dimegin [2,4-di-(alpha-methoxyethyl)-deuteroporphyrin IX] was investigated with respect to cellular uptake, intracellular localization and cell survival following photodynamic treatment in human cell lines derived from the skin (SCL1 and SCL2, squamous cell carcinoma; HaCaT keratinocytes; N1 fibroblasts). Using flow cytometry, we determined the cellular fluorescence as a marker of the uptake of Dimegin after incubation for 24 h. The intracellular localization of Dimegin was analysed using fluorescence microscopy and co-staining with fluorescent dyes specific for cell organelles. Following irradiation with an incoherent light source (580-740 nm) using a light dose of 24 J/cm2, phototoxicity was determined by means of trypan blue dye exclusion, MTT assays and growth curves. The relative Dimegin fluorescence of the different cell lines declined as follows: SCL1 > HaCaT > N1 > SCL2. Intracellular localization of Dimegin was found in the mitochondria. For all cell lines Dimegin concentrations above 15 microM yielded a significant phototoxic effect. The EC50 for SCL1 cells was 8.9 +/- 2.0 microM Dimegin. The EC50 for the cell lines increased as follows: SCL1 < HaCaT < N1 < SCL2, thus correlating with the cellular fluorescence of Dimegin. The results of the MTT assay were confirmed by trypan blue dye exclusion assay and growth curves. In conclusion, the study shows that Dimegin is an effective photosensitizer with a rapid mechanism of action in vitro, resulting in an immediate loss of plasma membrane integrity following irradiation.     

Effects of chronic ethanol exposure on oxidation and NMDA-stimulated neuronal death in primary cortical neuronal cultures

Two enzymes of detoxification were studied in blood samples from 27 patients with ulcerative colitis (UC) and 18 controls to determine whether there is an abnormality in sulfur metabolism in UC. Thiol methyltransferase (TMT) activity was measured in erythrocyte membranes as the extent of conversion of 2-mercaptoethanol to S-methyl-2-mercaptoethanol with [3H]methyl-S-adenosyl methionine as methyl donor. Phenol sulfotransferase (PST) activity was measured in platelet homogenates as the extent of sulfation of p-nitrophenol with 3-phosphoadenosine 5-phospho[35S]sulfate (PAPS) as sulfate donor. TMT activity was significantly higher in UC patients (27.0 vs 17.1 nmol/mg protein/hr; P < 0.005). No difference in PST activity was found. We conclude that TMT may be up-regulated in UC to detoxify excess hydrogen sulfide exposed to the peripheral blood compartment. This may arise from either increased luminal sulfide production or reduced colonic detoxification.     

Enzymology of mitomycin C metabolic activation in tumour tissue. Characterization of a novel mitochondrial reductase

In this study, the enzymology of mitomycin C (MMC) bioactivation in two murine colon adenocarcinomas, MAC 16 and MAC 26, was examined. Subcellular quinone reductase assessment via cytochrome c reduction confirmed a number of active enzymes. MAC 16 exhibited 22-fold greater levels of cytosolic DT-diaphorase than MAC 26, while microsomal NADPH:cytochrome P-450 reductase levels were similar in both tumour types. Metabolism of MMC by subcellular fractions isolated from both MAC 16 and MAC 26 was quantitated by monitoring the formation of the principle metabolite 2,7-diaminomitosene (2,7-DM) via high-performance liquid chromatography (HPLC). In MAC 16 only, activity displaying the properties of cytosolic DT-diaphorase and microsomal NADPH:cytochrome P-450 reductase was detected and confirmed, using the enzyme inhibitors dicoumarol and cytochrome P-450 reductase antiserum, respectively. The highest level of MMC metabolism was associated with the mitochondrial fraction from both tumours and was the sole enzyme activity detected in MAC 26. The greatest mitochondrial drug metabolism was achieved in the presence of NADPH as cofactor and hypoxia (MAC 16-specific activity, 3.67 +/- 0.58 nmol/30 min/mg; MAC 26 specific-activity, 3.87 +/- 0.71 nmol/30 min/mg) and was unaffected by the addition of the inhibitors dicoumarol and cytochrome P-450 reductase antiserum. NADH-dependent mitochondrial activity was only observed in MAC 16 at approximately 4-fold less than that seen with NADPH. MAC 26 homogenate incubations displayed enhanced metabolism under hypoxia, presumably due to the presence of the identified mitochondrial enzyme. MAC 16 homogenates showed no increase in metabolism under hypoxia, suggesting that other enzyme(s) may be predominant. These data indicate the presence of a novel mitochondrial one-electron reductase capable of metabolising MMC in MAC 16 and MAC 26.