Detection of virulence factors in culturable Escherichia coli isolates from water samples by DNA probes and recovery of toxin-bearing strains in minimal o-nitrophenol-beta-D-galactopyranoside-4-methylumbelliferyl-beta-D-g luc uronide media. A total of 449 Escherichia coli isolates in treated and raw water sources were submitted to DNA-DNA hybridization using seven different DNA probes to detect homology to sequences that code for Shiga-like toxins I and II; heat-stabile and heat-labile toxins, adherence factors EAF and eae, and the fimbrial antigen of entero-hemorrhagic E. coli. Fifty-nine (13%) of the isolates demonstrated homology with one or more specific DNA probes. More than 50% of the isolates in treated water were not recovered in MMO-4-methylumbelliferyl-beta-D-glucuronide media designed for detection of this indicator.


A new assay for lignin-type peroxidases employing the dye azure B. The discovery in 1983 of fungal "ligninases" capable of catalyzing the peroxidation of nonphenolic aromatic lignin components has been seen as a major advance in understanding how certain basidiomycete fungi can completely degrade lignin. The ability of these lignin-type peroxidases to covert millimolar concentrations of veratryl alcohol to veratraldehyde, indicated by a change in the A310 of veratraldehyde, has become the standard assay for routine quantitation of LP activity. A new assay based on the oxidation of micromolar concentrations of the dye Azure B is presented. Although it is as simple and rapid as the veratryl alcohol assay, it appears to overcome some of the shortcomings of that assay. In particular, interference from UV- and short-wavelength visible-light-absorbing materials is greatly reduced and assay specificity is improved.


Enhanced biodegradation of phenanthrene in oil tar-contaminated soils supplemented with Phanerochaete chrysosporium. In recent years, the white rot fungus Phanerochaete chrysosporium has shown promise as an organism suitable for the breakdown of a broad spectrum of environmental pollutants, including polynuclear aromatic hydrocarbons (PAHs). The focus of this study was to determine whether P. chrysosporium could effectively operate in an actual field sample of oil tar-contaminated soil. The soil was loaded with [14C]phenanthrene to serve as a model compound representative of the PAHs. Soil with the native flora present under static, aerobic conditions with buffering (pH 5.0 to 5.5) displayed full mineralization on the order of 20% in 21 days. The addition of P. chrysosporium was synergistic, with full mineralization on the order of 38% in 21 days. In addition to full mineralization, there was an increase in the proportion of radiolabelled polar extractives, both soluble and bound, in the presence of P. chrysosporium. From this study, it is apparent that the native soil microflora can be prompted into full mineralization of PAHs in some contaminated soils and that this mineralization can be enhanced when supplemented with the white rot fungus P. chrysosporium. With further refinement, this system may prove an effective bioremediation technology for soils contaminated with PAHs.


Rates of inactivation of waterborne coliphages by monochloramine. A sophisticated water quality monitoring program was established to evaluate virus removal through Denver's 1-million-gal (ca. 4-million-liter)/day Direct Potable Reuse Demonstration Plant. As a comparison point for the reuse demonstration plant, Denver's main water treatment facility was also monitored for coliphage organisms. Through the routine monitoring of the main plant, it was discovered that coliphage organisms were escaping the water treatment processes. Monochloramine residuals and contact times (CT values) required to achieve 99% inactivation were determined for coliphage organisms entering and leaving this conventional water treatment plant. The coliphage tested in the effluent waters had higher CT values on the average than those of the influent waters. CT values established for some of these coliphages suggest that monochloramine alone is not capable of removing 2 orders of magnitude of these specific organisms in a typical water treatment facility. Electron micrographs revealed one distinct type of phage capable of escaping the water treatment processes and three distinct types of phages in all.


Characterization of transcription initiation and termination signals of the proteinase genes of Lactococcus lactis Wg2 and enhancement of proteolysis in L. lactis. The transcription initiation signals of the prtP and prtM genes specifying the proteolytic activity of Lactococcus lactis subsp. cremoris Wg2 were mapped by primer extension. The strength of these promoters was analyzed with promoter-screening vector pGKV410, and they appeared to be weaker than previously isolated promoters of strain Wg2. In addition, a putative transcription terminator downstream of the prtP gene was characterized by using the terminator-screening vector pGKV259. The putative terminator decreased the transcription activity of lactococcal promoter P59 by approximately 70% in both Bacillus subtilis and L. lactis. Deletion of a part of the stem-loop structure of the terminator decreased the negative effect on transcription, indicating that the structure could indeed function as a terminator of transcription. The proteolytic activity of the lactococcal host was enhanced by placing the originally oppositely oriented prt genes in tandem and replacing the relatively weak promoters upstream of the prt genes with the stronger promoter, P32, from the chromosome of L. lactis Wg2.


Metabolic and energetic control of Pseudomonas mendocina growth during transitions from aerobic to oxygen-limited conditions in chemostat cultures. Several metabolic fluxes were analyzed during gradual transitions from aerobic to oxygen-limited conditions in chemostat cultures of Pseudomonas mendocina growing in synthetic medium at a dilution rate of 0.25 h-1. P. mendocina growth was glucose limited at high oxygen partial pressures (70 and 20% pO2) and exhibited an oxidative type of metabolism characterized by respiratory quotient (RQ) values of 1.0. A similar RQ value was obtained at low pO2 (2%), and detectable levels of acetic, formic, and lactic acids were determined in the extracellular medium. RQs of 0.9 +/- 0.12 were found at 70% pO2 for growth rates ranging from 0.025 to 0.5 h-1. At high pO2, the control coefficients of oxygen on catabolic fluxes were 0.19 and 0.22 for O2 uptake and CO2 production, respectively. At low pO2 (2%), the catabolic and anabolic fluxes were highly controlled by oxygen. P. mendocina showed a mixed-type fermentative metabolism when nitrogen was flushed into chemostat cultures. Ethanol and acetic, lactic, and formic acids were excreted and represented 7.5% of the total carbon recovered. Approximately 50% of the carbon was found as uronic acids in the extracellular medium. Physiological studies were performed under microaerophilic conditions (nitrogen flushing) in continuous cultures for a wide range of growth rates (0.03 to 0.5 h-1). A cell population, able to exhibit a near-maximum theoretical yield of ATP (YmaxATP = 25 g/mol) with a number of ATP molecules formed during the transfer of an electron towards oxygen along the respiration chain (P/O ratio) of 3, appears to have adapted to microaerophilic conditions.(ABSTRACT TRUNCATED AT 250 WORDS)


Evidence of hydrolytic route for anaerobic cyanide degradation. Products observed during anaerobic cyanide transformation are consistent with a hydrolytic pathway (HCN + H2O <--> HCONH2 + H2O <--> HCOOH + NH3). Formate, the most frequently observed product, was generally converted to bicarbonate. Formamide was rapidly hydrolyzed to formate upon exposure to the anaerobic consortium but was not detected as an intermediate of cyanide transformation.


Identification of geosmin as a volatile metabolite of Penicillium expansum. Cultures of Penicillium expansum produce a musty, earthy odor. Geosmin [1,10-trans-dimethyl-trans(9)-decalol] was identified by gas chromatography-mass spectrometry from headspace samples of P. expansum cultures. Olfactory comparison of P. expansum cultures with a geosmin standard indicated geosmin is the primary component of the odor associated with P. expansum.


Evaluation of the ability of primary selective enrichment to resuscitate heat-injured and freeze-injured Listeria monocytogenes cells. Resuscitation rates of injured Listeria monocytogenes on conventional selective Listeria enrichment broth and nonselective Trypticase soy broth containing 0.6% yeast extract were compared. Cells were heated to 60 degrees C for 5 min or frozen at -20 degrees C for 7 days. Inoculation of Trypticase soy broth-yeast extract with the stressed cells resulted in growth that was superior to that in Listeria enrichment broth. Injured cells were fully recovered at 6 to 8 h.


A simple method to test condoms for penetration by viruses. A method by which virus penetration through condoms can be tested with simple, inexpensive equipment is described. The method uses chi X174 bacteriophage as the challenge virus and physiologically relevant pressure. Penetration by 0.1 microliters (or less) of challenge suspension can be readily detected. As examples, latex and natural-membrane condoms were examined.


Plasmids in Listeria monocytogenes in relation to cadmium resistance. One hundred and seventy-three unrelated Listeria monocytogenes strains isolated from humans, animals, the environment, and food were analyzed for the presence of plasmids. Extrachromosomal DNA was found in 28% of the strains. Plasmid DNA was extracted more frequently from L. monocytogenes serogroup 1 strains (35%) than from serogroup 4 strains (15%). Among strains from food and the environment, 40% and 29%, respectively, harbored plasmids, whereas only 13% of the strains from humans and animals with listeriosis bore plasmids. We also investigated the susceptibility of 90 strains to seven antibiotics and four heavy-metal salts. No antibiotic resistance could be detected, but 95.3% of the plasmid-positive strains and only 12.7% of the plasmid-negative strains were resistant to cadmium. The plasmid-determined genetic basis of cadmium resistance was proven by conjugation between strains of L. monocytogenes and by cure of the plasmid. This is the first time that plasmids of L. monocytogenes have been shown to be associated with cadmium resistance.


Rapid determination of members of the family Enterobacteriaceae in drinking water by an immunological assay using a monoclonal antibody against enterobacterial common antigen. An immunological method for the detection of members of the family Enterobacteriaceae in drinking water was developed. The method was based on a sandwich enzyme-linked immunosorbent assay (ELISA) with monoclonal antibody immunoglobulin G2a 898 against enterobacterial common antigen. The enterobacterial common antigen sandwich ELISA combined with selective preenrichment culture could be performed in only 24 h. Six hundred sixty-eight water samples from a variety of German public water supplies were screened to verify the effectiveness of the new method. Ninety-eight percent of the results obtained by the immunological method could be confirmed by conventional microbiological methods. The immunological method proved to be considerably faster and more specific and sensitive than the standard method specified by the German drinking water regulations.


Metabolic by-products of anaerobic toluene degradation by sulfate-reducing enrichment cultures. Two dead-end metabolites of anaerobic toluene transformation, benzylsuccinic acid and benzylfumaric acid, accumulated in sulfate-reducing enrichment cultures that were fed toluene as the sole carbon source. Stable isotope-labeled toluene and gas chromatography-mass spectrometry were used to confirm that the compounds resulted from toluene metabolism. The two metabolites constituted less than 10% of the toluene carbon (over 80% was mineralized to carbon dioxide, according to a previous study). This study demonstrates that the novel nonproductive pathway proposed by Evans and coworkers (P. J. Evans, W. Ling, B. Goldschmidt, E. R. Ritter, and L. Y. Young, Appl. Environ. Microbiol. 58:496-501, 1992) for a denitrifying pure culture applies to disparate anaerobic bacteria.


Influence of 2,4,6-trinitrotoluene (TNT) concentration on the degradation of TNT in explosive-contaminated soils by the white rot fungus Phanerochaete chrysosporium. The ability of Phanerochaete chrysosporium to bioremediate TNT (2,4,6-trinitrotoluene) in a soil containing 12,000 ppm of TNT and the explosives RDX (hexahydro-1,3,5-trinitro-1,3,5- triazine; 3,000 ppm) and HMX (octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine; 300 ppm) was investigated. The fungus did not grow in malt extract broth containing more than 0.02% (wt/vol; 24 ppm of TNT) soil. Pure TNT or explosives extracted from the soil were degraded by P. chrysosporium spore-inoculated cultures at TNT concentrations of up to 20 ppm. Mycelium-inoculated cultures degraded 100 ppm of TNT, but further growth was inhibited above 20 ppm. In malt extract broth, spore-inoculated cultures mineralized 10% of added [14C]TNT (5 ppm) in 27 days at 37 degrees C. No mineralization occurred during [14C]TNT biotransformation by mycelium-inoculated cultures, although the TNT was transformed.


Comparison of selected methods for the enumeration of fecal coliforms and Escherichia coli in shellfish. In a comparison of five selected methods for the enumeration of fecal coliforms and Escherichia coli in naturally contaminated and sewage-seeded mussels (Choromytilus spp.) and oysters (Ostrea spp.), a spread-plate procedure with mFC agar without rosolic acid and preincubation proved the method of choice for routine quality assessment.


Purification and characterization of dihydrobenzophenanthridine oxidase from elicited Sanguinaria canadensis cell cultures. Upon treatment of Papaveracea cells with fungal elicitors, the biosynthesis of benzo[c]phenanthridine alkaloids is induced. Dihydrobenzophenanthridine oxidase, which catalyzes a later step in the biogenesis of these alkaloids, is one of the enzymes whose activity is elevated in the process. Here we report the 211-fold purification of the oxidase from elicited Sanguinaria canadensis by a combination of ammonium sulfate fractionation, DEAE-Sephadex, CM-Sephadex, Sephadex G-200, and either phenyl Superose or gel filtration chromatography. The purified enzyme utilized molecular oxygen to oxidize dihydrosanguinarine to sanguinarine with concomitant formation of hydrogen peroxide. A pH optimum of 7.0, Vmax of 27 nkat/mg protein, and apparent Km of 6.0 microM for dihydrosanguinarine were determined. Dihydrochelerythrine was also found to be a substrate for the purified enzyme, displaying an apparent Km of 10 microM. However, neither dihydronorsanguinarine nor the indole alkaloid ajmalicine was oxidized, indicating that the enzyme has some substrate specificity. Apparent molecular weight estimates by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the most purified enzyme preparation obtained contained a major component of 77 kDa and two minor components between 59 and 67 kDa that can be associated with oxidase activity. Purified enzyme preparations possessed activity that was inhibited by sodium diethyldithiocarbamate, sodium azide, potassium cyanide, 1,4-DL-dithiothreitol, and mercaptoethanol.


Reactions of thrombin-serpin complexes with thrombospondin. Activated platelets release proteins that form stable complexes with thrombin (J. J. Miller, P. C. Browne, and T. C. Detwiler, Biochem. Biophys. Res. Commun. 151, 9-15, 1988). A working model for the reaction (P. C. Browne, J. J. Miller, and T. C. Detwiler, Arch. Biochem. Biophys. 265, 534-538, 1988) includes a dissociable complex of thrombin with released platelet protease nexin, leading to formation of a nondissociable thrombin-nexin complex that then becomes disulfide linked to thrombospondin. This disulfide-linked complex is converted back to the thrombin-nexin complex by reduction of disulfide bonds. Results that allow elaboration on this model are presented. After longer periods of incubation or after incubation with higher concentrations of thrombin, the amount of thrombin complexed with thrombospondin exceeded the amount of thrombin-nexin complex recovered after reduction of disulfide bonds. When the reaction mixture included inhibitors of formation of the thrombin-nexin complex, a slow formation of the thrombin-thrombospondin complex was observed. It was concluded that there is a nexin-independent as well as the faster nexin-dependent disulfide linkage of thrombin to thrombospondin. Addition of thrombin-antithrombin III complexes to the supernatant solution of activated platelets also led to complexes with thrombospondin, demonstrating that serpins other than platelet protease nexin facilitate incorporation of thrombin into complexes with thrombospondin. By heparin affinity chromatography, it was shown that thrombin-nexin complexes dissociably associate with thrombospondin prior to formation of disulfide-linked complexes. These observations are incorporated into a more detailed model of the reaction.


Monomaleimidogold labeling of the gamma subunit of the Escherichia coli F1 ATPase examined by cryoelectron microscopy. A novel approach for locating sites of interest in a protein complex has been developed using monomaleimidonanogold (MMN). The Escherichia coli F1 ATPase, when prepared without the delta subunit, contains only a single reactive cysteine on one of the three copies of the alpha subunit. This site was reacted with MMN and the gold cluster visualized on the protein complex by cryoelectron microscopy. Additional sites for modification with MMN were added by introducing cysteine residues through site-directed mutagenesis. Labeling of two mutants, gamma S8-C and gamma T106-C, in which Ser8 and Thr106, respectively, had been replaced by a cysteine, placed the gold cluster on the central mass that is seen in the hexagonal projection of the ECF1 complex. The results establish that the central mass contains the N-terminal part of the gamma subunit.


Post-transcriptional regulation of mouse renal cytochrome P450 2E1 by testosterone. Our previous studies demonstrated that the sex-related difference in renal metabolism of N-nitrosodimethylamine in C3H/HeJ mouse was due to the sexual dichotomy of cytochrome P450 2E1 (P450 2E1) and that renal P450 2E1 in female mouse was inducible by testosterone. The present study demonstrates that the sex-related difference in renal P450 2E1 and the testosterone-mediated regulation also occurred in other mouse strains studied. The time- and dose-responses in the testosterone-mediated regulation of P450 2E1 were characterized. 19-Nortestosterone, an analog of testosterone, was shown to have an effect on the regulation of mouse renal P450 2E1 similar to that of testosterone. Testicular feminized mice (Tfm, a mouse strain devoid of functional androgen receptors) had about only one-tenth the renal P450 2E1 mRNA level as the wild-type male mice and testosterone treatment of the Tfm mice had no effect on the level of renal P450 2E1 mRNA. The result suggests that androgen receptor plays an important role in the sex- and testosterone-related regulation of mouse renal P450 2E1. To study the mechanism of the sex-related and testosterone-mediated regulation of P450 2E1 mRNA in mouse kidney, the transcription rate of the P450 2E1 gene was measured by a nuclear run-on transcription assay. Although the kidneys of male and testosterone-treated female mice had much higher steady-state levels of P450 2E1 mRNA, their transcription rates of the P450 2E1 gene were not higher than the kidneys of untreated female mice. This result suggests that the sex- and testosterone-related regulation of mouse renal P450 2E1 is predominantly at the post-transcriptional level.


Interaction of mitochondrially bound rat brain hexokinase with intramitochondrial compartments of ATP generated by oxidative phosphorylation and creatine kinase. Previous work led to the conclusion that, during oxidative phosphorylation, mitochondrially bound hexokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1) from rat brain was dependent on intramitochondrially compartmented ATP as substrate. The present study demonstrated that, when oxidative phosphorylation was functioning concurrently, mitochondrial creatine kinase could also generate intramitochondrial ATP serving as substrate for hexokinase. In the absence of concurrent oxidative phosphorylation, the kinetics of glucose phosphorylation with ATP generated by creatine kinase were not consistent with the supply of ATP from a saturable intramitochondrial compartment as formed during oxidative phosphorylation. Evidence for intramitochondrially compartmented ATP, generated by creatine kinase, was obtained; this was distinct from compartmented ATP generated by oxidative phosphorylation in terms of kinetics of generation of the compartment and its capacity, sensitivity to release by carboxyatractyloside, and sensitivity to disruption by digitonin. That oxidative phosphorylation did induce a dependence on intramitochondrial ATP as a substrate was further indicated by the observation that, although the initial rate of glucose phosphorylation by mitochondrial hexokinase depended on the extramitochondrial concentration of ATP present at the time oxidative phosphorylation was initiated, a final steady state rate of glucose phosphorylation was attained that was independent of extramitochondrial ATP levels. These and previous results emphasize the probable importance of nucleotide compartmentation in regulation of cerebral glycolytic and oxidative metabolism.


Effects of ligands on reduction of oxygen by vanadium(IV) and vanadium(III). V(IV) and V(III) reduce molecular oxygen with increasing rates as the pH is raised from 6.0 to 7.4. Under all conditions tested, V(IV) is the more efficient reductant. EDTA and ATP generally inhibit the reduction of oxygen by V(III) and V(IV). In contrast, desferrioxamine accelerates the reduction of oxygen by V(IV) but with decreasing effectiveness at pH 7.4 compared to pH 6.0, while desferrioxamine accelerates the reduction of oxygen by V(III) only at pH 6.0. Histidine enhances the reduction of oxygen by V(IV) at pH 7.0 and 7.4. The observed rates of oxygen reduction by V(III) and V(IV) imply that the intracellular distribution of vanadium among its redox states reflects not an equilibrium but a steady state.


Inactivation of Escherichia coli 2-amino-3-ketobutyrate CoA ligase by phenylglyoxal and identification of an active-site arginine peptide. Treatment of homogeneous preparations of 2-amino-3-ketobutyrate CoA ligase from Escherichia coli, a pyridoxal 5'-phosphate-dependent enzyme, with phenylglyoxal, 4-(oxyacetyl)phenoxyacetic acid, 2,3-butanedione, or 1,2-cyclohexanedione results in a time- and concentration-dependent loss of enzymatic activity. Phenylglyoxal in 50 mM phosphate buffer (pH 7.0) is the most effective modifier, causing > 95% inactivation within 20 min at 25 degrees C. Controls establish that this inactivation is not due to modifier-induced dissociation or photoinduced nonspecific alteration of the ligase. The substrate, acetyl CoA, or the coenzyme, pyridoxal 5'-phosphate, gives > 50% protection against inactivation. Enzyme partially inactivated by phenylglyoxal has the same Km value for glycine but the Vmax decreases in proportion to the observed level of inactivation. Whereas the native apoligase shows good recovery of activity with time in parallel with an increase in 428-nm absorptivity when incubated with pyridoxal 5'-phosphate, no such effects are seen with the phenylglyoxal-modified apoligase. Reaction of the enzyme with [14C]phenylglyoxal allowed for the isolation of a peptide which, by amino acid composition and sequencing data, was found to correspond to residues 349-378 in the intact enzyme. These results indicate that arginine residue-366 and/or residue-368 in the primary structure of E. coli 2-amino-3-ketobutyrate ligase is at the active site.


Catalytic properties of the human cytochrome P450 2E1 produced by cDNA expression in mammalian cells. A full-length cDNA encoding human cytochrome P450 2E1 was expressed in mammalian cell lines using the vaccinia virus expression system. Immunoblot analysis showed that the expressed protein reacted with a polyclonal antibody against rat 2E1 and comigrated with P450 2E1 from human liver microsomes. P450 2E1 expressed in Hep G2 cells, a human cell line which contains both cytochrome b5 and NADPH:P450 oxidoreductase, was able to metabolize several known P450 2E1 substrates: N-nitrosodimethylamine (NDMA), N-nitrosomethylbenzylamine (NMBzA), p-nitrophenol, phenol, and acetaminophen. Apparent Km and Vmax values for NDMA demethylation were 22 microM and 173 pmol/min/mg microsomal protein, respectively. P450 2E1 expressed in TK-143 cells, which do not contain b5, displayed Km and Vmax values of 31 microM and 34 pmol/min/mg microsomal protein, respectively. Incorporation of purified rat liver b5 into TK-143 microsomes increased the Vmax 2.2-fold and decreased the Km to 22 microM. Addition of b5 to Hep G2 microsomes resulted in a 1.6-fold increase in Vmax, but showed no effect on the Km. P450 2E1 expressed in Hep G2 cells was shown to metabolize NMBzA with a Km of 47 microM and Vmax of 213 pmol/min/mg microsomal protein. Addition of b5 lowered the Km to 27 microM, but had no effect on Vmax. These results demonstrate conclusively that P450 2E1 is responsible for the low Km forms of NDMA demethylase and NMBzA debenzylase observed in liver microsomes and that these activities are affected by cytochrome b5.


Hormonal regulation of rat renal cytochrome P450s by androgen and the pituitary. The hormonal regulation of rat renal cytochrome P450s, P450 4A2 (K-5) and K-2, was investigated. The level of P450 4A2 in male rats was five times that in female rats and accounted for some 90% of total cytochrome P450, measured photometrically. Lauric acid omega- and (omega-1)-hydroxylation activities of renal microsomes of male rats were also higher than those of female rats. The sex differences in lauric acid hydroxylation activity seemed to arise from the differences in P450 4A2 concentrations, according to an immunochemical study. P450 K-2 was a female-dominant form in rat kidneys. The level of P450 K-2 in renal microsomes of male rats was one-tenth that of P450 4A2. Castration of male rats decreased the levels of P450 4A2 and treatment of castrated male rats with testosterone reversed the decrease. The castration of male rats decreased the lauric acid hydroxylation of the renal microsomes to the level of female rats. The administration of testosterone to castrated male rats reversed the decrease. Hypophysectomy of male rats decreased the level of P450 4A2 and the administration of growth hormone reversed the decrease when intermittent injections mimicking the male secretory pattern were given, although continuous administration mimicking the female secretory pattern did not. Castration of male rats did not affect the level of P450 K-2, but testosterone decreased its level. Hypophysectomy of male rats increased the level of P450 K-2 and growth hormone decreased its level in hypophysectomized rats. These results suggested that the expression of P450 4A2 was regulated by androgen or growth hormone and regulation of P450 4A2 was different from that of P450 K-2. To explore the regulation of renal cytochrome P450 further, testosterone was given to control (intact) or hypophysectomized adult female rats. P450 4A2 was induced in the kidneys of both control and hypophysectomized female rats to close to the level of male rats. Thus, P450 4A2 was directly regulated by testosterone as well as growth hormone, and the regulation of the male-dominant form in rat kidneys was different from that of the male-specific form in the rat liver, which is regulated mostly by growth hormone.


Direct electrochemistry of thioredoxins and glutathione at a lipid bilayer-modified electrode. By using direct electrochemical analysis we have established that the reduction of Escherichia coli thioredoxin (EcT), T4 thioredoxin (T4T), and glutathione (GSSG) occurs at a self-assembled lipid bilayer-modified gold electrode via two separate one-electron processes. The first electron transfer has half-wave potentials of -0.05 +/- 0.01, -0.07 +/- 0.01, and -0.06 +/- 0.01 V, whereas the second one has values of -0.48 +/- 0.01, -0.39 +/- 0.01, and -0.45 +/- 0.01 V, for EcT, T4T, and GSSG, respectively. The scan-rate dependence of the cyclic voltammetry indicates, for both waves, that the process of electron transfer is dominated by a bulk diffusion of free species to and from the electrode, and that strongly adsorbed species do not significantly contribute at the scan rates used. The voltage separation of the peak currents indicates a quasi-reversible electron transfer process with an electrochemical rate constant which is larger for the second (lower potential) electron than for the first one. Using the above half-wave potentials of the one-electron steps, one can calculate a thermodynamic half-wave potential for the two-electron reduction processes. The values of these potentials are -0.265, -0.23, and -0.25 V for EcT, T4T, and GSSG, respectively. These are in excellent agreement with literature values obtained from equilibrium measurements of enzyme-catalyzed reactions involving these species. It is quite clear from these results that lipid bilayer-modified electrodes provide a biocompatible and direct means of efficiently carrying out electrochemical reactions with sulfur-based redox systems, as we have previously shown to be the case with metalloproteins.


Human mitochondrial aldehyde dehydrogenase substrate specificity: comparison of esterase with dehydrogenase reaction. Substrate specificity of human mitochondrial low Km aldehyde dehydrogenase (EC 1.2.1.3) E2 isozyme has been investigated employing p-nitrophenyl esters of acyl groups of two to six carbon atoms and comparing with that of aldehydes of one to eight carbon atoms. The esterase reaction was studied under three conditions: in the absence of coenzyme, in the presence of NAD (1 mM), and in the presence of NADH (160 microM). The maximal velocity of the esterase reaction with p-nitrophenyl acetate and propionate as substrates in the presence of NAD was 3.9-4.7 times faster than that of the dehydrogenase reaction. Under all other conditions the velocities of dehydrogenase and esterase reactions were similar; the lowest kcat was for p-nitrophenyl butyrate in the presence of NAD. Stimulation of esterase activity by coenzymes was confined to esters of short acyl chain length; with longer acyl chain lengths or increased bulkiness (p-nitrophenyl guanidinobenzoate) no effect or even inhibition was observed. Comparison of kinetic constants for esters demonstrates that p-nitrophenyl butyrate is the worst substrate of all esters tested, suggesting that the active site topography is uniquely unfavorable for p-nitrophenyl butyrate. This fact is, however, not reflected in kinetic constants for butyraldehyde, which is a good substrate. The substrate specificity profile as determined by comparison of kcat/Km ratios was found to be quite different for aldehydes and esters. For aldehydes kcat/Km ratios increased with the increase of chain length; with esters under all three conditions, a V-shaped curve was produced with a minimum at p-nitrophenyl butyrate.


Effects of temperature acclimation on the expression of hepatic cytochrome P4501A mRNA and protein in the fish Fundulus heteroclitus. Previous studies showed that hydrocarbon induction of hepatic microsomal monooxygenase activity is attenuated in the teleost fish Fundulus heteroclitus acclimated to low temperature. The basis of that attenuation, and the effects of temperature on monooxygenase activity, were examined by analyzing liver cytochrome P4501A (CYP1A) mRNA, protein, and catalytic activity in control and beta-naphthoflavone (BNF)-treated F. heteroclitus acclimated to 6 or 16 degrees C. There were no temperature-related differences in total P450 content, NADPH-cytochrome c (P450) reductase activity, ethoxyresorufin O-deethylase (EROD) activity, or immunoquantified CYP1A content in hepatic microsomes of untreated fish. Fish acclimated to 16 degrees C and given a single intraperitoneal injection of BNF exhibited a rapid rise and fall in CYP1A mRNA content and an induction of EROD activity and CYP1A protein that was undiminished over 7 days. Similarly treated fish acclimated at 6 degrees C showed an increase in CYP1A mRNA content greater than that in 16 degrees C fish, but with no significant increase in EROD activity or CYP1A content over 7 days. Examined over a longer term, microsomal EROD activity was significantly induced by BNF in fish at both temperatures; activity peaked at 5-7 days in 16 degrees C fish, while in 6 degrees C fish the activity continued to rise slowly over 25 days. However, the greatest activity reached in 6 degrees C fish (0.68 nmol/min/mg) was less than half that seen in the warmer animals (1.46 nmol/min/mg). Immunodetectable CYP1A content showed the same trend as EROD activity, and the turnover number (nmol product formed/min/nmol CYP1A) for EROD activity was about the same in all groups, indicating that concentration of the catalyst alone could account for the different patterns of microsomal activity. CYP1A mRNA content was again induced to a similar degree by BNF in both the 6 and the 16 degrees C fish; the apparent half-life of the mRNA was substantially longer in cold-acclimated than in warm-acclimated BNF-treated fish. Comparing the levels of CYP1A mRNA and protein at the two acclimation temperatures following BNF treatment indicates that translational activity, rather than transcriptional activity, is the sensitive point in the effect of temperature on CYP1A induction in these fish.


Ethanol and oleate inhibition of alpha-ketoisovalerate and 3-hydroxyisobutyrate metabolism by isolated hepatocytes. Ethanol inhibited glucose synthesis from alpha-ketoisovalerate by isolated rat hepatocytes without significant inhibition of flux through the branched-chain alpha-ketoacid dehydrogenase complex. Accumulation of 3-hydroxyisobutyrate, an intermediate in the catabolism of alpha-ketoisovalerate, was increased by ethanol, indicating inhibition of flux at the level of 3-hydroxyisobutyrate dehydrogenase. 3-Hydroxybutyrate caused the same effects as ethanol, suggesting inhibition was a consequence of an increase in the mitochondrial NADH/NAD+ ratio. Flux through the 3-hydroxyisobutyrate dehydrogenase was more sensitive to regulation by the mitochondrial NADH/NAD+ ratio than flux through the branched-chain alpha-ketoacid dehydrogenase. Oleate also inhibited glucose synthesis from alpha-ketoisovalerate, but marked inhibition of flux through the branched-chain alpha-ketoacid dehydrogenase complex was caused by this substrate.


Biosynthesis of monoterpenes: inhibition of (+)-pinene and (-)-pinene cyclases by thia and aza analogs of the 4R- and 4S-alpha-terpinyl carbocation. (+)-Pinene cyclase (synthase) from Salvia officinalis leaf catalyzes the cyclization of geranyl pyrophosphate, via (3R)-linalyl pyrophosphate and the (4R)-alpha-terpinyl cation, to (+)-alpha-pinene and to lesser quantities of stereochemically related monoterpene olefins, whereas (-)-pinene cyclase converts the same achiral precursor, via (3S)-linalyl pyrophosphate and the (4S)-alpha-terpinyl cation, to (-)-alpha-pinene and (-)-beta-pinene and to lesser amounts of related olefins. Racemic thia analogs of the linalyl and alpha-terpinyl carbocation intermediates of the reaction sequence were previously shown to be good uncompetitive inhibitors of monoterpene cyclases, and inhibition was synergized by the presence of inorganic pyrophosphate. These results suggested that the normal reaction proceeds through a series of carbocation:pyrophosphate anion paired intermediates. Both the (4R)- and the (4S)-thia and -aza analogs of the alpha-terpinyl cation were prepared and tested as inhibitors with the antipodal pinene cyclases, both in the absence and in the presence of inorganic pyrophosphate. Although the inhibition kinetics were complex, cooperative binding of the analogs and inorganic pyrophosphate was demonstrated, consistent with ion pairing of intermediates in the course of the normal reaction. Based on the antipodal reactions catalyzed by the pinene cyclases, stereochemical differentiation between the (4R)- and the (4S)-analogs was anticipated; however, neither enzyme effectively distinguished between enantiomers of the thia and aza analogs of the alpha-terpinyl carbocation. Enantioselectivity in the enzymatic conversion of (RS)-alpha-terpinyl pyrophosphate to limonene by the pinene cyclases was also examined. Consistent with the results obtained with the thia and aza analogs, the pinene cyclases were unable to discriminate between enantiomers of alpha-terpinyl pyrophosphate in this unusual reaction. Either the alpha-terpinyl antipodes are too similar to allow differentiation by the pinene cyclases, or these enzymes lack an inherent requirement to distinguish the (4R)- and (4S)-forms because they encounter only one enantiomer in the course of the normal reaction from geranyl pyrophosphate.


Evidence for an essential histidine residue in 4S-limonene synthase and other terpene cyclases. (4S)-Limonene synthase, isolated from glandular trichome secretory cell preparations of Mentha x piperita (peppermint) leaves, catalyzes the metal ion-dependent cyclization of geranyl pyrophosphate, via 3S-linalyl pyrophosphate, to (-)-(4S)-limonene as the principal product. Treatment of this terpene cyclase with the histidine-directed reagent diethyl pyrocarbonate at a concentration of 0.25 mM resulted in 50% loss of enzyme activity, and this activity could be completely restored by treatment of the preparation with 5 mM hydroxylamine. Inhibition with diethyl pyrocarbonate was distinguished from inhibition with thiol-directed reagents by protection studies with histidine and cysteine carried out at varying pH. Inactivation of the cyclase by dye-sensitized photooxidation in the presence of rose bengal gave further indication of the presence of a readily modified histidine residue. Protection of the enzyme against inhibition with diethyl pyrocarbonate was afforded by the substrate geranyl pyrophosphate in the presence of Mn2+, and by the sulfonium ion analog of the linalyl carbocation intermediate of the reaction in the presence of inorganic pyrophosphate plus Mn2+, suggesting that an essential histidine residue is located at or near the active site. Similar studies on the inhibition of other monoterpene and sesquiterpene cyclases with diethyl pyrocarbonate suggest that a histidine residue (or residues) may play an important role in catalysis by this class of enzymes.


Sublethal oxidant stress induces a reversible increase in intracellular calcium dependent on NAD(P)H oxidation in rat alveolar macrophages. A concentration-dependent elevation of intracellular calcium ([Ca2+]i) and oxidation of NAD(P)H occurred in alveolar macrophages during exposure to sublethal tert-butylhydroperoxide concentrations (tBOOH) (< or = 100 microM in 1 ml with 1 x 10(6) cells). Oxidation of NAD(P)H preceded a rise in [Ca2+]i. The elevation of [Ca2+]i was reversible at < 50 microM tBOOH exposure and the return to the steady state [Ca2+]i correlated temporally with repletion of NAD(P)H. At > 50 microM tBOOH, the changes in NAD(P)H and [Ca2+]i were sustained. The relative contributions of NADPH and NADH oxidation were examined by varying the substrates supplying reducing equivalents and by inhibiting glutathione reductase activity. The results suggested that at < 50 microM tBOOH, oxidation of NADPH predominated, while at > 50 microM tBOOH, NADH oxidation predominated. A complex relationship between the relative roles of NADPH and NADH oxidation and the elevation of [Ca2+]i was revealed: (i) reversible oxidation of NADPH is associated with the initial and reversible elevation of [Ca2+]i at < 50 microM tBOOH; (ii) the sustained elevation of [Ca2+]i at > 50 microM tBOOH correlates with the sustained oxidation of NADH; and (iii) the changes in [Ca2+]i did not depend on influx of extracellular Ca2+. We speculate that at low tBOOH, Ca2+ was released from the NADPH/NADP(+)-sensitive mitochondrial Ca2+ pool while higher tBOOH caused additional Ca2+ release from GSH/GSSG-sensitive nonmitochondrial Ca2+ pools with sustained elevation of [Ca2+]i due to decreased mitochondrial Ca2+ reuptake.


Regulation of sex pheromone biosynthesis in the housefly, Musca domestica: relative contribution of the elongation and reductive steps. The regulation of production of the sex pheromone (Z)-9-tricosene (Z9-23:Hy) in the housefly, Musca domestica, was studied by examining the chain length specificity of the fatty acyl-CoA elongation reactions and the reductive conversion of fatty acyl-CoAs to alkenes in 1- and 4-day-old male and female houseflies. Microsomal preparations from 4-day-old female insects produced as the predominant alkene Z9-23:Hy when incubated with malonyl-CoA, NADPH, and [9,10-3H2]oleoyl-CoA (18:1-CoA), whereas microsomal preparations from 4-day-old male insects produced predominantly (Z)-9-heptacosene (Z9-27:Hy). These are the major alkenes produced in vivo by Day 4 females and males, respectively. Microsomes prepared from both Day 1 males and Day 1 females produced Z9-27:Hy as the major alkene from labeled 18:1-CoA. This is the major alkene produced in vivo by both sexes at Day 1. An examination of the chain length specificity of the elongation reactions showed that microsomes prepared from Day 4 male insects readily elongated both 18:1-CoA and 15-[15,16-3H2]tetracosenoyl-CoA (24:1-CoA) to 28-carbon moieties, whereas microsomes from Day 4 female insects did not efficiently elongate either substrate beyond 24 carbons. With high substrate concentrations, microsomes prepared from male insects converted 24:1-CoA to Z9-23:Hy more efficiently than did those from females, whereas under lower and presumably more physiological substrate concentrations, microsomes from females had slightly higher activity than did those from males. Taken together, these data show that the regulation of the chain length of the alkenes, and thus sex pheromone production, in the housefly resides predominantly in the elongation reactions and not in the step which converts the fatty acyl-CoA to hydrocarbon.


Differentiation of HL-60 cells by phorbol ester is correlated with up-regulation of protein kinase C-alpha. To clarify the mechanism of 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced macrophage-like differentiation of HL-60 cells, we investigated the correlation between the effects of protein kinase C (PKC) inhibitors on the induction of markers of TPA-induced differentiation and those on suggested critical steps of the differentiation. H-7, sphingosine, and trifluoroperazine significantly suppressed TPA-induced cell adhesion but their effects on the induction of acid phosphatase and nonspecific esterase differed among the inhibitors. The three inhibitors failed to affect on TPA-induced annexin I expression. In contrast, staurosporine markedly suppressed the induction of all these markers. The effects of the inhibitors on some suggested critical steps of the differentiation, a rapid phosphorylation of specific proteins, a rapid membrane association of PKC, and down-regulation of PKC at 18 h after addition of TPA, were not correlated with those on the differentiation marker induction. Only the effect of the inhibitors on up-regulation of PKC-alpha was closely correlated with TPA-induced annexin I expression; staurosporine inhibited up-regulation of PKC-alpha but other inhibitors did not similarly affect the induction of annexin I expression. These results suggest that PKC-alpha is intimately related to macrophage-like differentiation of HL-60 cells by TPA.


Differential effects of iron and inflammation on ferritin synthesis on free and membrane-bound polyribosomes of rat liver. We have examined the distribution of ferritin mRNA to free and endoplasmic reticulum (ER)-bound liver polyribosomes during inflammation and iron treatment of rats. Postnuclear tissue supernatants were fractionated on a discontinuous sucrose gradient developed to separate free and bound polyribosomes. Total RNA recovered averaged 3.2 mg/g tissue, 40% of which was with ER and 30% with the free polyribosomes, about 25% being with the postribosomal/RNP fraction. Slot-blot hybridization of equal portions of RNA revealed that 12 h after injection of turpentine to induce inflammation, ferritin mRNA was concentrated on the ER-bound polyribosomes, while it was concentrated on the free polyribosomes 2 h after injection of ferric ammonium citrate. Differences were highly significant, based on multiple determinations and densitometry. Profiles of ferritin mRNA distribution on linear sucrose gradients corroborated the differential findings. Concentrations of total ferritin mRNA per gram liver doubled with iron treatment but were not significantly different 12 h after turpentine treatment. At the same time point after turpentine, ferritin protein synthesis was increased twofold, as measured by the 1 h incorporation of [14C]leucine. We conclude that a significant portion of ferritin mRNA always associates with the ER-bound polyribosomes, and that inflammation and iron differentially alter the polysomal distribution of ferritin mRNA, suggesting that two different kinds of mRNA may be involved.


Mechanism of activation of the NAD-malic enzyme from Ascaris suum by fumarate. The mechanism of activation of the NAD-malic enzyme from Ascaris suum by fumarate has been probed using initial velocity studies, deuterium isotope effects, and isotope partitioning of the E:Mg:malate complex. Fumarate exerts its activating effect by decreasing the off-rate for malate from the E:Mg:malate and E:NAD:Mg:malate complexes. Fumarate is a positive heterotropic effector of the NAD-malic enzyme at low concentrations (K act approximately 0.05 mM) and an inhibitor competitive against malate (Ki approximately 25 mM). The activation by fumarate results in a decrease in the Ki malate and an increase in V/K malate of about 2-fold, while the maximum velocity remains constant. Isotope partitioning studies of E:Mg:[14C]malate indicate that the presence of fumarate results in a decrease in the malate off-rate constant by about 2.2-fold. The deuterium isotope effects on V and V/K malate are both 1.6 +/- 0.1 in the absence of fumarate, while in the presence of 0.5 mM fumarate DV is 1.6 +/- 0.1 and D(V/K malate) is 1.1 +/- 0.1. These data are also consistent with a decrease in the off-rate for malate from E:NAD:Mg:malate, resulting in an increase in the forward commitment factor for malate and manifested as a lower value for D(V/K malate). There is a discrimination between active and activator sites for the binding of dicarboxylic acids, with the activator site preferring the extended configuration of 4-carbon dicarboxylic acids, while the active site prefers a configuration in which the 4-carboxyl is twisted out of the C1-C3 plane. The physiologic importance and regulatory properties of fumarate in the parasite are also discussed.


Lec15 cells transfer glucosylated oligosaccharides to protein. B4-2-1 cells (Lec15 cells) are Chinese hamster ovary cells deficient in mannosylphosphoryldolichol synthase activity. They synthesize the truncated lipid intermediate Man5GlcNAc2-P-P-dolichol rather than the Glc3Man9GlcNAc2-P-P-dolichol synthesized by wild-type cells. In this report we present evidence that these cells did synthesize glucosylated Man5GlcNAc2-P-P-dolichol, but this species represented only a minor fraction of the labeled oligosaccharide-lipid. On the other hand, glucosylated oligosaccharides were a major species transferred to protein in these cells, showing that in vivo, glucosylated oligosaccharides are preferentially transferred to protein. The truncated oligosaccharides found in B4-2-1 cells were removed from the protein by N-glycanase treatment, since they were resistant to both endo-beta-N-acetylglucosaminidase H and F activity. B4-2-1 cells processed the glucosylated, truncated oligosaccharides transferred to G protein of vesicular stomatitis virus, leading to infectious virus.


Glutathione S-transferases of human lung: characterization and evaluation of the protective role of the alpha-class isozymes against lipid peroxidation. Glutathione S-transferase (GST) isozymes of human lung have been purified, characterized, quantitated, and, based on their structural and immunological profiles, identified with their respective classes. The tau-, mu-, and alpha-class GSTs represented 94, 3, and 3% activities of total human lung GSTs toward CDNB, respectively, and 60, 10, and 30% of total GST protein, respectively. Both the mu- and the alpha-class GSTs of human lung exhibited heterogeneity. The two mu-class GSTs of human lung had pI values of 6.5 and 6.25 and were differentially expressed in humans. Significant differences were seen between the kinetic properties of these two isozymes and also between the lung and liver mu-class GSTs. The alpha-class GST isozymes of lung resolved into three peaks during isoelectric focusing corresponding to pI values of 9.2, 8.95, and 8.8. All three alpha-class GSTs isozymes had blocked N-termini and were immunologically similar to human liver alpha-class GSTs. Peptide fingerprints generated by SV-8 protease digestion and CNBr cleavage indicated minor structural differences between the liver and the lung alpha-class GSTs. The three alpha-class GSTs of lung expressed glutathione peroxidase activities toward the hydroperoxides of phosphatidylcholine, phosphatidylethanolamine, and phosphatidylglycerol, with Km values in the range of 22 to 87 microM and Vmax values in the range of 67-120 mol/mol/min, indicating the involvement of the alpha-class GSTs in the protection mechanisms against peroxidation. All three classes of lung GSTs expressed activities toward leukotriene A4 methyl ester and epoxy stearic acid but the mu-class GSTs had relatively higher activities toward these substrates.


Molecular cloning of the chicken gizzard telokin gene and cDNA. Telokin is a protein which consiste of the C-terminal portion of smooth muscle myosin light chain kinase (MLCK) (M. Ito, R. Dabrowska, V. Guerriero, Jr., and D. J. Hartshone (1989) J. Biol. Chem. 264, 13971-13974). In this study, the chicken gizzard telokin cDNA and gene were cloned and analyzed. The telokin cDNA coded 157 amino acid residues which were completely identical to the C-terminal portion of the amino acid sequence of chicken gizzard MLCK. The telokin gene was coded in a 6.3-kb EcoRI genomic fragment and it consisted of three exons. The 5'-leader sequence of the telokin cDNA and genomic sequence revealed that the telokin gene was included in the MLCK gene and the transcription started in the intronic sequence of the MLCK gene. The analysis of the telokin gene suggests that the telokin expression was under the control of an independent promotor. Northern blotting and the reverse transcriptase-polymerase chain reaction methods revealed that telokin was expressed not only in chicken gizzard but also in chicken heart, lung, intestine, and skeletal muscle although the levels of the expression in the latter were much less than that in the gizzard.


Tissue-specific expression, induction, and inhibition through metabolic intermediate-complex formation of guinea pig cytochrome P450 belonging to the CYP2B subfamily. The tissue-specific expression and induction of P450GP-1, a constitutive form of cytochrome P450 of the guinea pig classified into the CYP2B subfamily, were studied. Prior to these studies, a P450 form (P450GP-1 PB) was purified from phenobarbital-treated guinea pigs and the properties were compared with those of the P450GP-1. This form was judged to be the same as P450GP-1 existing in untreated animals by comparisons of their N-terminal amino acid sequences, peptide maps, and affinities toward anti-P450GP-1 antibody. Immunostaining of P450GP-1 revealed that the lung and small intestine as well as the liver of untreated guinea pigs contain P450GP-1, while none or only small amounts of this P450 form were observed in the kidney, heart, spleen, urinary bladder, and testis. The amount of liver P450GP-1 protein expressed in untreated guinea pigs was estimated to be 19.4% of the total cytochrome P450 and this form was increased 1.7-fold by phenobarbital treatment. Similarly, intestinal P450GP-1 was increased by phenobarbital treatment. However, lung P450GP-1 was not increased by the treatment. It was also observed that the liver P450GP-1 is induced with SKF-525A to the same extent as with phenobarbital. On the other hand, dexamethsone, p,p'-dichlorodiphenyltrichloroethane, and isosafrole showed no or only a weak ability to increase the liver P450GP-1 content. The drug-metabolizing activities in the liver microsomes of SKF-525A-pretreated guinea pigs were lower than those in phenobarbital-treated animals, although the P450GP-1 protein was induced equally by these treatments. The low activities of SKF-525A-treated animals in the drug metabolisms were attributed to the formation of the metabolic-intermediate complex between P450GP-1 and SKF-525A metabolite. These results permitted us to conclude that the tissue specificity in the expression of guinea pig P450 belonging to the CYP2B subfamily and the inducibility with chemicals are similar to those of rat CYP2B1, although the constitutive expression of guinea pig liver P450GP-1 is much higher than that of CYP2B1.


Binding of pulmonary surfactant protein A to galactosylceramide and asialo-GM2. The binding of pulmonary surfactant protein A (SP-A) to glycolipids was examined in the present study. The direct binding of SP-A on a thin-layer chromatogram was visualized using 125I-SP-A as a probe. 125I-SP-A bound to galactosylceramide and asialo-GM2, but failed to exhibit significant binding to GM1, GM2, asialo-GM1, sulfatide, and Forssman antigen. The study of 125I-SP-A binding to glycolipids coated onto microtiter wells also revealed that SP-A bound to galactosylceramide and asialo-GM2. SP-A bound to galactosylceramides with non-hydroxy or hydroxy fatty acids, but showed no binding to either glucosylceramide or galactosylsphingosine. Excess native SP-A competed with 125I-SP-A for the binding to asialo-GM2 and galactosylceramide. Specific antibody to rat SP-A inhibited 125I-SP-A binding to glycolipids. In spite of chelation of Ca2+ with EDTA or EGTA, SP-A retained a significant binding to glycolipids. Inclusion of excess monosaccharides in the binding buffer reduced the glycolipid binding of SP-A, but failed to achieve complete abolishment. The oligosaccharide isolated from asialo-GM2 is also effective at reducing 125I-SP-A binding to the solid-phase asialo-GM2. From these data, we conclude that SP-A binds to galactosylceramide and asialo-GM2, and that both saccharide and ceramide moieties in the glycolipid molecule are important for the binding of SP-A to glycolipids.


Synthesis of high-affinity, hydrophobic monosaccharide derivatives and study of their interaction with concanavalin A, the pea, the lentil, and fava bean lectins. Concanavalin A (Con A) and agglutinins from the pea (PSA), lentil (LCH), and fava bean (VFA) constitute a group of D-mannose/D-glucose binding legume lectins. In addition to their sugar binding specificity, these lectins also contain sites that bind hydrophobic ligands. The present study explores a class of nonpolar binding sites reportedly present adjacent to the carbohydrate binding site in PSA, LCH, and VFA. A series of 2-O- and 3-O-substituted nitrobenzoyl and nitrobenzyl derivatives of methyl alpha-D-glucopyranoside and methyl alpha-D-mannopyranoside were synthesized. Evaluation of their binding to Con A, PSA, LCH, and VFA was carried out by the technique of hapten inhibition of precipitation reaction. The hapten inhibition assay results reveal that the presence of a methyl or methylene group at the O-2 or O-3 position of the sugar is essential for hydrophobic interaction with PSA, LCH, and VFA. The substitution of methyl by nitrobenzyl leads to enhanced binding (1.7-16.7 times for the 2-O-substituted compounds and 7.9-40.5 times for the 3-O-substituted compounds) with the m-nitrobenzyl group contributing to maximum binding. A hydrophobic interaction is also involved between Con A and 2-O-nitrobenzyl derivatives, resulting in enhanced binding, but the corresponding 3-O-isomers bind poorly due probably to steric reasons. These results may be rationalized on the basis of the recently published X-ray data of Con A and VFA. The nitrobenzyl derivatives, after transformation to their azido analogs, have potential applications in the photoaffinity labeling of these lectins.


Effects of cationic polypeptides on the activity, substrate interaction, and autophosphorylation of casein kinase II: a study with calmodulin. The effects of basic polypeptides on the ability of casein kinase II to phosphorylate an exogenous substrate (calmodulin) are correlated with steady-state autophosphorylation of the alpha- and beta-subunits of casein kinase II. Polylysine and polyarginine increase autophosphorylation of the alpha-subunit with a concomitant decrease in beta-subunit phosphorylation, while enhancing casein kinase II-stimulated phosphorylation of calmodulin over 100-fold. The highly basic carboxyl terminal segment of the endogenous p21c-Ki-ras has similar effects on the phosphorylation of calmodulin and the alpha- and beta-subunits of casein kinase II. Altering the concentration of cationic polypeptides produces a biphasic effect on the phosphorylation of both calmodulin and the alpha-subunit, which correlate positively with each other but do not correlate with beta-subunit phosphorylation. When the KCl concentration is changed, casein kinase II activity correlates positively only with alpha-subunit phosphorylation. In contrast, the biphasic response of calmodulin phosphorylation by casein kinase II at different Ca2+ concentrations correlates positively with both alpha- and beta-subunit phosphorylation. Therefore, in the presence of basic protein activators, the rate of phosphorylation of a substrate, calmodulin, correlates with steady-state phosphorylation of the alpha-subunit, but not with the beta-subunit under all conditions tested. Endogenous cationic factors may modulate the in vivo activity of casein kinase II and alter the interaction of the enzyme with specific intracellular substrates.


Identification of arginyl residues involved in the binding of ferredoxin-NADP+ reductase from Anabaena sp. PCC 7119 to its substrates. Ferredoxin-NADP+ reductase from the cyanobacterium Anabaena sp. PCC 7119 was chemically modified by the alpha-dicarbonyl reagent phenylglyoxal. The studies of the inactivation by this compound, which is specific for arginyl residues, of both the diaphorase and NADPH-cytochrome c reductase activities, characteristic of the enzyme, are indicative of the involvement of at least one group of this kind in the binding site of NADP+ and a second one implicated in the interaction with ferredoxin. After specific cleavage of a FNR sample incubated with [7-14C]phenylglyoxal, two major labeled peptides were identified. The peptide which exhibited the higher degree of modification corresponded to residues 208-242. It contained four arginine residues but only two of them were the target of the modification: Arg224 and Arg233. Protection studies with protein substrates and sequence comparison with other reductases allow us to propose that these residues in Anabaena sp. PCC 7119 FNR must be involved in the interaction with the pyridine nucleotide. The second peptide corresponds to residues 75-103 and although it contains three arginine residues, Arg77 is the only one that exhibits the modification. This residue seems to be a key one in the interaction of this reductase with ferredoxin.


The MgATPase activity of rat cardiac sarcoplasmic reticulum is a function of the calcium ATPase protein. Magnesium-dependent ATPase (MgATPase) activity is associated with many E1-E2 or P-type transport ATPases including the sarcoplasmic reticulum (SR) calcium ATPase. The SR isolated from rat heart has a MgATPase activity which is 6-12 times faster than the MgATPase activity of the SR isolated from dog heart. To determine the origin of the high MgATPase activity of rat heart SR, we compared and contrasted cardiac SR isolated from both species. The preparations were similar in the following ways: (i) contamination by other organelles; (ii) the comigration of MgATPase activity with calcium-dependent ATPase (CaATPase) activity through a sucrose gradient; (iii) a similar ATPase activity sensitivity to pH and ATP concentration; (iv) the high and similar of sensitivity of ATPase activity to detergent; and (v) a similar protein profile. In both preparations, a single protein in the 105,000-Da region of polyacrylamide gels was phosphorylated by ATP, and the phosphorylated species was an acylphosphate formed in the presence and absence of calcium. Dimethyl sulfoxide, which slows acylphosphoenzyme breakdown, markedly inhibited both CaATPase and MgATPase activities of both preparations but not other enzyme activities. Importantly, the specific inhibitor of the SR calcium pump, thapsigargin, completely inhibited the CaATPase activity with an I50 of 6-7 nM; however, a higher concentration (I50 of 2 microM) was required to inhibit the MgATPase activity of the rat cardiac SR. These results provide evidence that the MgATPase activity of rat cardiac SR is part of the enzyme cycle of the calcium ATPase protein.


Effects of mutations at residue 309 of the large subunit of ribulosebisphosphate carboxylase from Synechococcus PCC 6301. Previous studies [G. S. Hudson et al. (1989) J. Biol. Chem. 265, 808-814] showed that the faster turnover rates and lower affinities for CO2 of ribulosebisphosphate carboxylase/oxygenases from C4 plants, compared to C3 and C3/C4 plants, were specified by the chloroplast-encoded large subunits. In pairs of closely related C3 and C4 species from three genera, these kinetic changes were accompanied by only three to six amino acid residue substitutions, depending on the genus. None of these substitutions occurred near the active site and only one, 309Met (C3) to Ile (C4), was common to all three genera. Unlike the plant carboxylases, the highly homologous enzyme from the cyanobacterium Synechococcus PCC 6301 folds and assembles properly when its rbcL and rbcS genes are coexpressed in Escherichia coli. Furthermore, the cyanobacterial enzyme has Ile at position 309 of the large subunit, a high turnover number, and a poor affinity for CO2. 309Ile was replaced with Met and several other residues by site-directed mutagenesis of the cyanobacterial rbcL. Met and Leu were tolerated at this position with no alteration in the kinetic or structural properties of the assembled holoenzyme. However, substitution with Val, Gly, Trp, or Arg prevented the assembly of the subunits. The indifference to Met or Ile at this position, as well as the tolerance for Leu which is not observed with any natural ribulosebisphosphate carboxylase, leads to the conclusion that either the 309Met/Ile substitution has no effect on the kinetic properties of the plant enzyme, despite the correlation apparent in previous studies, or the cyanobacterial enzyme is sufficiently different from the plant enzyme in other respects that the influence of residue 309 is masked.


The interaction of Trolox C, a water-soluble vitamin E analog, with ferrylmyoglobin: reduction of the oxoferryl moiety. The oxidation of the heme iron of metmyoglobin by H2O2 yields an oxo ferryl complex (FeIV = O), similar to Compound II of peroxidases, as well as a protein radical; this high oxidation state of myoglobin is known as ferrylmyoglobin. The interaction of Trolox, a water-soluble vitamin E analog, with ferrylmyoglobin entailed two sequential one-electron oxidations of the phenolic antioxidant with intermediate formation of a phenoxyl radical and accumulation of a quinone end product. These oxidation reactions were linked to individual reductions of ferrylmyoglobin to metmyoglobin, as indicated by the value of the relationship [metmyoglobin]formed/[Trolox]consumed: 1.92 +/- 0.28. The Trolox-mediated reduction of ferrylmyoglobin to metmyoglobin could proceed directly, i.e., electron transfer from the phenolic-OH group in Trolox to the oxoferryl moiety, or indirectly, i.e., sequential electron transfer from Trolox to a protein radical to the oxoferryl moiety. The former mechanism is supported by the finding that the high oxidation heme iron is reduced under conditions where the tyrosyl residues are blocked by o-acetylation and when hemin is substituted for myoglobin. The latter mechanism is consistent with the following observations: (a) the EPR signal ascribed to the protein radical is suppressed by Trolox, with the concomitant appearance of the EPR spectrum of the Trolox phenoxyl radical and (b) the rate of ferrylmyoglobin reduction by Trolox is decreased with increasing number of tyrosyl residues in the proteins of horse myoglobin (titrated by o-acetylation) and sperm whale myoglobin. The apparent discrepancy between these observations can be reconciled by considering that both electrophilic centers in ferrylmyoglobin--the oxoferryl heme moiety and the protein radical--function independently of each other and that recovery of ferrylmyoglobin by Trolox could be effected through the tyrosyl residues, albeit at slower rates. The mechanistic aspects of these results are discussed in terms of the two main redox transitions in the myoglobin molecule encompassing valence changes of the heme iron and electron transfer of the tyrosyl residue in the protein and linked to the two sequential one-electron oxidations of Trolox.


Activation of protein kinase C isozymes by contractile stimuli in arterial smooth muscle. Protein kinase C (PKC) has been proposed to be involved in the regulation of vascular smooth muscle (VSM) contractile activity. However, little is known in detail about the activation of this kinase or specific isozymes of this kinase by contractile stimuli in VSM. As an index of PKC activation, Ca(2+)- and phospholipid-dependent histone IIIS kinase activity was measured in the particulate fraction from individual strips of isometrically contracting carotid arterial smooth muscle. Phorbol 12,13-dibutyrate (PDB) increased PKC activity in the particulate fraction (155% over resting value by 15 min) with a time course which paralleled or preceded force development. Stimulation with the agonist histamine (10(-5) M) resulted in rapid increases in both force and particulate fraction PKC activity which was maximal by 2 min (increase of 139%) and partially sustained over 45 min (increase of 41%). KCl (109 mM), which evokes a sustained contractile response, caused a slow increase (124% by 45 min) in particulate fraction PKC activity. No significant increases in activator-independent histone kinase activity were observed in response to any stimulus tested. PKC alpha and PKC beta were identified as the principal Ca2+/phospholipid-dependent PKC isozymes expressed in this tissue. In unstimulated arterial tissue, the ratio of immunodetectable isozyme content (alpha:beta) was estimated to be 1:1 in the particulate and 1.5:1 in the cytosolic fractions. Upon stimulation with each of the three contractile stimuli, particulate fraction PKC content assessed by immunoblotting increased with a time course and to an extent comparable to the observed changes in PKC activity. There was no evidence of differential regulation of the PKC alpha or -beta isozymes by PDB compared to the other contractile stimuli. These results indicate that diverse contractile stimuli are capable of tonically activating PKC in preparations of functional smooth muscle, and are consistent with a functional role for PKC alpha and/or -beta in the regulation of normal smooth muscle contractile activity.


Antioxidant effect of manganese.

Antioxidant effect of manganese. The antioxidant effects of manganese and other transition metals were studied as the inhibition of microsomal lipid peroxidation and crocin bleaching by peroxyl radicals. The peroxyl radical scavenging capacity was measured by competition kinetics analysis. While Zn(II), Ni(II), and Fe(II) were almost completely ineffective, Mn(II) and Co(II) showed a free radical scavenging capacity, exhibiting relative rate constant ratios respectively of 0.513 and 0.287. This indicates that Mn(II) is by far the most active. Therefore, the chain-breaking antioxidant capacity of Mn(II) seems to be related to the rapid quenching of peroxyl radicals according to the reaction R-OO. + Mn(II) + H(+)-->ROOH+Mn(III). The antioxidant mechanism is discussed considering the different reduction potentials of the examined cations.


The specificity and elastinolytic activities of bovine cathepsins S and H. Cathepsins S and H were purified from bovine spleen and their catalytic properties compared. The enzymes were shown to be similar by chromatographic properties and by the ability to hydrolyze Bz-Phe-Val-Arg-NHMec. They could however be distinguished by the fact that cathepsin S reacted with Z-[125I]Tyr-Ala-CHN2 and hydrolyzed Z-Phe-Arg-NHMec whereas cathepsin H did not. The substrate and inhibitor specificities of cathepsin H suggest that unlike cathepsins B, L, and S, it cannot accommodate peptides with aromatic side chains in P2. Cathepsins L and S can accommodate the aromatic side chain of tyrosine in P1 readily, whereas cathepsins H and B cannot. The specificities of each enzyme for synthetic substrates and inhibitors have enabled the construction of models of the architecture of the active sites of the mammalian cysteine proteinases which clearly show the differences between the four enzymes. A significant characteristic of cathepsin S is that it can hydrolyze insoluble elastin at both acidic and neutral pH; this distinguishes it from all of the other lysosomal proteinases.


Studies on the holoenzyme biogenesis of the spinach ferredoxin-NADP+ reductase. An expression plasmid, pPreFNR, in which the DNA sequence coding for the spinach ferredoxin-NADP+ reductase precursor was under the control of prokaryotic transcription and translation initiation signals has been constructed. The plasmid directed the synthesis in Escherichia coli of a 43-kDa immunoreactive polypeptide which could be identified with the reductase preprotein. Analyses of bacterial extracts showed that the precursor was unstable and devoid of catalytic activities, suggesting that the presence of the transit peptide would not allow the assembly in E. coli of an active preholoenzyme. Furthermore, the reductase precursor was found to undergo a processing in E. coli. The proteolysed form, which retained 13 of the 55 residues of the transit peptide, was active, suggesting that removal of the first 42 residues of the presequence enabled the protein to properly fold and to bind the FAD prosthetic group in the bacterial host, as it was previously shown in the case of the mature form of the spinach reductase.


Akazara scallop troponin C: Ca(2+)-induced conformational change and interaction with rabbit troponin subunits. The number of specific Ca2+ bound to Akazara scallop troponin C was estimated to be 0.7 with an apparent binding constant of 5 x 10(5) M-1 (T. Ojima and K. Nishita, 1986, J. Biol. Chem. 261, 16749-16754). In the present paper, we report on the Ca(2+)-induced conformational changes in the troponin C and the interaction of the troponin C with rabbit troponin subunits. The Ca2+ binding to the troponin C caused a marked change in difference uv absorption spectra and a retardation of elution on Sephacryl S-200 gel filtration. However, its circular dichroism spectrum was hardly changed by the Ca2+ binding. These results suggest that the Ca2+ binding to the troponin C induced changes predominantly in tertiary structure rather than in secondary structure. Akazara scallop troponin C was shown to be able to bind to rabbit troponin I-Cellulofine affinity column, but the affinity was not greatly increased by Ca2+ unlike the case of rabbit troponin C. On hybridizing with rabbit troponin T and I, Akazara scallop troponin C was shown to be incapable of substituting rabbit troponin C; i.e., the hybrid troponin strongly inhibited the Mg-ATPase activity of rabbit actomyosin-tropomyosin irrespective of the presence or absence of Ca2+, thus recovering no Ca2+ sensitivity.


Crystal structure of peroxynitrite-modified bovine Cu,Zn superoxide dismutase. The crystal structure of bovine Cu,Zn superoxide dismutase modified with peroxynitrite (ONOO-) was determined by X-ray diffraction, utilizing the existing three-dimensional model of the native structure deposited in the Brookhaven Protein Data Bank (J. A. Tainer et al., J. Mol. Biol. 160, 181-217, 1982). The native structure and the modified derivative were refined to R factors of 19.0 and 18.7% respectively using diffraction data from 6.0 to 2.5 A. The major result after reaction with peroxynitrite was the appearance of electron density 1.45 A from a single epsilon carbon of Tyr-108, the only tyrosine residue in the sequence. Tyr-108 is a solvent-exposed residue 18 A from the copper atom in the active site. The electron density was consistent with nitration of Tyr-108 at one of the epsilon carbons to form 3-nitrotyrosine. We propose that the nitration occurs in solution by transfer of a nitronium-like species from the active site on one superoxide dismutase dimer to the Tyr-108 of a second dimer.


Binding of cytosolic aconitase to the iron responsive element of porcine mitochondrial aconitase mRNA. The 5' end of porcine mitochondrial aconitase mRNA contains an iron responsive element (IRE)-like secondary structure (T. Dandekar, R. Stripecke, N. K. Gray, B. Goosen, A. Constable, H. E. Johansson, and M. W. Hentze (1991) EMBO J. 10, 1903-1909). A protein from a liver extract binds to a mitochondrial aconitase RNA probe and supports the identification of this sequence as an IRE. Purified cytosolic aconitase but not the mitochondrial enzyme binds to this IRE as well as to a ferritin IRE. All forms of cytosolic aconitase, [4Fe-4S] enzyme, [3Fe-4S] enzyme and apoenzyme bind with similar affinity. A Kd of 0.25 nM was calculated for the apoaconitase-IRE interaction from Scatchard analysis. These results support the conclusion that cytosolic aconitase is an IRE-binding protein which may regulate translation of mitochondrial aconitase mRNA.


CA074 methyl ester: a proinhibitor for intracellular cathepsin B. The specificity of compound CA074 [N-(L-3-trans-propylcarbamoyloxirane-2-carbonyl)-L-isoleucyl-L-pro line] for the inactivation of cathepsin B was quantified in in vitro measurements with cysteine endopeptidases from cattle, it being found that the compound is a very rapid inactivator of cathepsin B (rate constant 112,000 M-1.s-1), with barely detectable action on cathepsins H, L, and S or m-calpain. Conversion of the proline carboxyl group of the inhibitor to the methyl ester virtually abolished the effect on cathepsin B, and a possible explanation for the importance of the carboxyl is presented on the basis of the tertiary structure of cathepsin B. It was found that CA074 methyl ester (1 microM, 3 h) caused selective inactivation of the intracellular cathepsin B of human gingival fibroblasts in culture, in contrast to other available agents, and we suggest that CA074 methyl ester will be of value in the elucidation of the biological functions of cathepsin B.


[Present status of gene diagnosis in cancer]. Advances in molecular genetics in the past decade enabled us to analyze the cause of mendelian disorders at molecular level and a variety of mutations, not only in point mutations and deletion in exons but also in those occurred in regulatory elements or in RNA processing have been precisely identified. Such a variety of mutations may constitute variable clinical manifestations even in the simple mendelian disorders. On the other hand, pathogenesis of common diseases is much complicated and remains greatly to be elucidated. However, if we could use the strategies applied in the past few years for mendelian disorders, it seems to be not difficult to approach them. It is recommended to categorize a certain disease into subgroups for distinguishing their heterogenous phenotypes by clinical, biochemical and other properties. Owing to the success in making a subgroup (FAB classification), many subtype-specific translocations were found in leukemia, and then, rearrangement of relevant genes is also being shown. The best example is seen in chronic myelocytic leukemia. Since rearrangement of ABL and BCR was shown and both genes were cloned, detection of minimal residual diseases after intensive treatment became possible at 10(-6) level using RT-PCR technique. Recently developed interphase cytogenetics using FISH has visualized Ph1 translocation in metaphase cells and also in round nuclei, suggesting a potential use in monitoring the effect of certain drugs during treatment. Furthermore, very selective targeting therapy is being devised using antisense DNA.


[New analogues of methotrexate].

[New analogues of methotrexate]. New antifolate antimetabolites are currently developed and three analogues of methotrexate (MTX) (Trimetrexate, Edatrexate, TNP-351) are undergoing clinical trials in Japan. Trimetrexate characteristically enters cells primarily by passive transport, which is different from the mechanism of MTX. The increased therapeutic efficacy of Edatrexate is expected, because of the higher concentration of polyglutamates than MTX in tumor cells. TNP-351 is a novel antifolate, which has a different chemical structure from other antifolates. The current state of clinical trials of three antifolates and the indications for chemotherapy of lung cancer are reviewed.


[Enhancement of the effects of anticancer agents on B16 melanoma cells by combination with cepharanthine--I. Alkaloids]. The anticancer effect of a podophyllotoxin derivative, etoposide (ETOP), and the vinca alkaloids, vincristine (VCR) and vindesine (VDS), on the colony forming efficiency (CFE) of B16 melanoma cells, and the CFE inhibition by the combination with a biscoclaurine alkaloid, Cepharanthine (Ceph) were studied in vitro. By combination with Ceph, CFE was reduced about 44% for ETOP, 31% for VCR and 30% for VDS compared to CFE without Ceph. Therefore, the effect of each anticancer agent was enhanced by combination with Ceph, especially that of Ceph and ETOP. The combination effect was greater when the low concentration of the agents was used. From the present study, the combination of ETOP and Ceph appears to be the more effective form of treatment.


[Enhancement of the effects of anticancer agents on B16 melanoma cells by combination with cepharanthine--II. Antimetabolite, alkylating agent, nitrosourea]. We have studied chemotherapy enhancement by the combination of the biscoclaurine alkaloid, Cepharanthine (Ceph), using the inhibition of colony forming efficiency (CFE) of B16 melanoma cells in vitro. Hydroxyurea, antimetabolite, showed a time-dependent inhibition of CFE, and the combination with Ceph enhanced CFE inhibition by 29%, which is stable and independent of the treatment time. Dacarbazine, a biological alkylating agent, given with Ceph showed a time-dependent inhibition of CFE (43%). MCNU, a nitrosourea, with Ceph showed the greatest inhibition of CFE (67%) among the anticancer agents used. The CFE inhibition was time-dependent and required a higher concentration of MCNU. In this series of studies using CFE inhibition of B16 melanoma cells in vitro, etoposide showed the best effect by the combination with Ceph, which seems to be a candidate for in vivo evaluation.


[Concentration of 5-fluorouracil in the blood and tissues of gastric and colo-rectal cancer patients after oral administration of UFT]. UFT is given to the patients with digestive cancer from the time before operation to prevent intra- and post-operative cancer dissemination and metastases. UFT (400 mg/day in terms of tegafur) was given preoperatively for 1-6 days in 6 patients with gastric cancer and 13 with colorectal cancer. The interval between the last administration and the beginning of the operation was 3.9 +/- 1.5 hours (mean +/- SD). The concentrations of tegafur, 5-FU, and uracil in the blood collected at the time of tumor resection were 9.68, 0.017, and 0.08 microgram/ml, respectively. In the patients with gastric cancer 5-FU concentration was 5.5 times higher in the normal mucosa, 3.3 times in lymph nodes, and 10.7 times in the tumor tissues than in the blood. In colorectal cancer patients, also, the 5-FU concentration was 5.6, 8.3 and 20.8 times higher in the normal mucosa, lymph nodes, and the tumor tissue, respectively, than in the blood. The 5-FU concentration in gastric cancer and colorectal cancer tissues decreased with time after administration of UFT but remained above the effective concentration 1.5-7 hours after administration of 200 mg. The tissue concentrations of FT-207, uracil, and 5-FU were correlated with each other.


[Blood concentrations of futraful, uracil and 5-fluorouracil (5-FU) after UFT administration in the various reconstructions after gastrectomy. Saitama UFT Research Group]. The study was undertaken in the total of 58 gastric cancer patients among which 17 of Billroth (BI), 14 of Billroth II (B II) anastomosis after subtotal gastrectomy, and 7 of jejunal interposition, 9 of double tract and 11 of Roux en Y anastomosis after total gastrectomy were included. Blood samples were taken before 200 mg of per oral UFT administration and after 1, 2, 3, 5 and 7hrs. consecutively. The blood Futraful (FT) level in the total gastrectomy groups reached peak concentration within 1hr and kept in relatively high level during the observation period of 7hrs. The time to maximum FT concentration delayed in almost of B I and a few of B II patients. The concentration curves of uracil (URA) and 5-FU were similar in shape, revealing steep increase and decrease except B I anastomosis which showed gentle course. The plotted maximum concentrations of URA and 5-FU in the every type of reconstruction showed a significant correlation in the regression line. In the analysis of AUC, URA/FT was under 10%, suggesting the longer retention of the unmetabolite type of FT and early disappearance of URA. The ratio of 5-FU/FT was indifferent in each reconstruction. 5-FU/URA was higher in subtotal rather than total gastrectomy groups. From the data obtained, blood concentration of 5-FU after UFT administration was considered to depend on the emptying status in the gastrectomies. And moreover, it depended on blood URA level, since FT from which 5-FU was derived, was kept still sufficiently remained during observation period.


[A study of urinary tegafur, 5-fluorouracil (5-FU) and uracil concentrations in the cases of gastric carcinoma for the confirmation of drug-taking compliance after UFT oral administration]. We have measured urinary tegafur (FT), 5-FU and uracil concentrations after UFT oral administration (300 mg daily for 7 days) to confirm drug-taking compliance in the 17 cases undergone gastrectomy. Urinary FT and 5-FU concentrations reached to the plateau 2 and 3 days after administration, respectively, and were maintained until the day after termination of administration. Subsequently, FT and 5-FU concentrations also decreased about 50% at 2 day, 20% at 3 day, 10% of the plateau values at 4 day after termination, respectively. The mean plateau value of urinary FT was 12.9 +/- 6.8 micrograms/dl, and that of urinary 5-FU was 0.67 +/- 0.50 microgram/dl. On the other hand, uracil concentration, was not different before and after administration because of the uracil being present endogenously. Therefore, it was suggested that measurement of urinary FT and 5-FU concentrations is useful for confirmation of UFT-taking compliance.


[Intraperitoneal administration of cisplatin (CDDP) for advanced or recurrent gastric cancer with peritoneal dissemination]. This study was undertaken in order to evaluate the effect of intraperitoneal administration of CDDP on fourteen patients with peritoneal dissemination of advanced or recurrent gastric cancer. The procedure was used together specific hydration transfusion and diuretics. Two patients showed a complete response and 5 patients a partial response. Following this therapy, 2 patients have survived for more than 6 months. Alimentary symptoms (nausea, vomiting) were found but renal toxicity and serious myelosuppression were not recognized in all patients.


[Clinical evaluation of intra-arterial methotrexate, vinblastine, doxorubicin and cis-platinum for bladder cancer]. Chemotherapy regimens including Methotrexate, Vinblastine. Doxorubicin and Cis-platinum (M-VAC) have shown objective responses in more than 50% of uroepithelial tumors. And intra-arterial chemotherapy can theoretically increase the drug concentration delivered to the tumor with maximum efficacy and less toxicity. In this report we review our experience at Matsuyama Red Cross Hospital between September 1987 and February 1991 using intra-arterial M-VAC in 21 evaluable patients with bladder cancer. Response was observed in 62% of the treated patients, two with complete remission and eleven with partial remission. WBC nadir and platelet nadir were mild to moderate. Other adverse effects such as mucositis, hair loss and creatinine elevation were seen in a small number of patients. Intra-arterial M-VAC can be one of the most useful forms of chemotherapy for patients with bladder cancer.


[Maintenance chemotherapy using UFT and FT for gynecological malignant tumors. The Gunma Prefectural Society for Study of Chemotherapy for Gynecological Cancer]. The Gunma Prefectural Society for Study of Chemotherapy for Gynecological Cancer made a comparative study on postoperative supplementary chemotherapy including UFT and FT-E for gynecological malignant tumors. The number of cases registered totaled 182, which was broken down into ovarian cancer 53 cases, uterine cervical cancer 89 cases and endometrial cancer 30 cases. Complete cases among them consisted of ovarian cancer 53 cases, uterine cervical cancer 86 cases and endometrial cancer 27 cases for a total of 166 cases. Ovarian cancer was stratified into the completely and incompletely removed cases, uterine cervical cancer into postoperative non-irradiated and irradiated cases and endometrial cancer into the positive metastasis (+) group and negative metastasis (-) group. Then the UFT administration group and FT-E administration group were compared for postoperative survival. There were control groups for both ovarian and endometrial cancer. UFT administration showed a significant improvement in the survival rate (P < 0.05) compared to the FT-E administration group in the cases of ovarian cancer completely removed and the cases of endometrial cancer irradiated after operation. No significant difference was observed between other groups compared. The incidence of side effects was 20.6% (20/97) in the UFT administration group and 3.6% (2/55) in the FT-E group. Neither group showed any serious side effects.


[PMUE therapy (CDDP, MMC, UFT, etoposide) for advanced gastric cancer--a case report]. CDDP, MMC, UFT and Etoposide (PMUE)-combined therapy was given to a 62-year-old man with advanced gastric carcinoma. PMUE therapy consists of i.v. injection of CDDP 75 mg/m2 and MMC 10 mg/body on day 1, i.v. injection of Etoposide 50 mg/body on days 3, 4 and 5 and consecutive daily administration of UFT 400 mg/body, with 3 weeks as one course. He was admitted for Borrmann type 3 gastric carcinoma with multiple liver metastasis, lymph node metastases and peritoneal dissemination, the underwent total gastrectomy with R2 lymph node dissection. He was treated four times with this therapy after sensitivity test for carcinostatic agents (SDI test), which resulted in complete remission, as confirmed by CT scan and second-look operation. The patient has currently been free of disease, and we conclude that this PMUE therapy is extremely effective for advanced gastric carcinoma.


[A case of gastric cancer with multiple liver metastases effectively treated with PMUE (CDDP, MMC, UFT, etoposide) hepatic arterial chemotherapy]. A 62-year-old male patient with progressive gastric cancer and multiple liver metastases (H3, P0, ss gamma, n4) underwent total gastrectomy (R1). After 2 years and 2 months, he was re-hospitalized with epigastric tumor caused by re-manifestation of liver metastasis as well as inappetence. Since a large focus of liver metastasis and intraportal tumor embolism was identified, a continuous intraarterial infusion tube utilizing Infuse-A-Port was inserted in the hepatic artery. After conducting 2 cycles of PMUE intra-arterial chemotherapy, the tumor size was reduced by 84% (PR); and CEA, which had been high upon rehospitalization, recovered to the normal level. After discharge, the patient has been receiving 5-FU arterial infusion as an outpatient and undergoing UFT oral chemotherapy. The efficacy has continued and he has been well for 3 years since operation. Often operations for gastric cancer accompanied with multiple liver metastasis meet with little success, and almost no case of prolonged survival has been reported. In this case, the effectiveness of PMUE arterial infusion chemotherapy was clear, the patient has been well for 3 years since operation, and is an interesting example with seemingly good prospects for long-survival.


[A five-year-survival case in which complete response was recognized after combined chemotherapy using 5-fluorouracil, adriamycin and mitomycin C (FAM) for unresectable gastric cancer]. FAM (5-fluorouracil, adriamycin, mitomycin C) therapy was performed on a 65-year-old man with unresectable gastric cancer. Cancer cells have not been recognized by endoscopic biopsy after the patient's complete response. He is alive without metastasis of recurrence for five years.


[Two cases of pulmonary metastasis of colorectal cancer successfully treated with oral 5'-DFUR]. Two patients with pulmonary metastasis of colorectal cancer, who had previously undergone radical resection of primary tumor, were orally administered 5'-DFUR at 1,200 mg per day. Complete responses were obtained in both. The duration of response was 42 and 13 weeks, respectively. Some side effects such as diarrhea and aguesia were found in each patient, but the treatment could be continued after temporary suspension for a while or by decreasing the dosage. We concluded that pulmonary metastasis of colorectal cancer can respond remarkably to oral administration of 5'-DFUR at 1,200 mg per day, if the tumor cells have sensitivity to this drug.


[A case report of bone metastasis diagnosed by MRI and effectively treated with UFT after breast conserving therapy for breast cancer]. A 66-year-old woman with left breast cancer (medullary carcinoma; T1cN1M0; Stage II A) was treated with breast conserving therapy combined with lumpectomy, radiotherapy, chemotherapy and endocrine therapy beginning in March, 1990. She complained of back pain and was diagnosed as having bone metastasis to the lower thoracic spine by bone scintigram and MRI examination in September, 1990. Oral administration of UFT (300 mg/day) was started, and 5 months later, back pain disappeared; nineteen months later, no definite findings of bone metastasis were observed on bone scintigram and MRI. It was concluded that long-term oral administration of UFT is an effective remedy for bone metastasis after breast conserving therapy for breast cancer.


[A case of advanced breast cancer with multiple organ metastases successfully treated by tamoxifen]. A 66-year-old postmenopausal woman presented in June 1991 with a giant ulcerated left breast tumor. She had discovered the tumor two years previously, but had never visited any medical institution. She was diagnosed as advanced breast cancer with multiple lung metastases, bone metastasis, and both supraclavicular lymph node metastases by physical examination, fine needle aspiration cytology, chest X-P, and bone scintigraphy. Incisional biopsy, performed to confirm the histological type of breast cancer and to evaluate estrogen and progesterone receptor (ER and PgR) status, revealed solid-tubular carcinoma. Both ER and PgR were highly positive at 322.6 and 228.0 fmol/mg protein, respectively. Therefore, endocrine therapy was chosen to treat this advanced breast cancer patient, although she had multiple organ metastases. Twenty mg of Tamoxifen a day was administered per os. After treatment with tamoxifen, the size of ulceration started to decreased and the dyspnea caused by multiple lung metastases was reduced. Eight weeks after, she showed partial response (PR) determined from the size of the ulceration and chest X-P. She has been maintaining PR for more than 9 months. Thus, Tamoxifen was shown to be very effective for this case of advanced breast cancer with multiple organ metastases.


[A case of jejunal vein thrombosis due to medroxyprogesterone acetate]. The patient was a 68-year-old woman with advanced breast cancer which had been treated by modified radical mastectomy two years and nine months earlier. After the surgery, tamoxifen citrate (TAM) was orally administered in addition to various types of chemotherapy. Because the patient complained of nausea and weight loss, medroxyprogesterone acetate (MPA) was orally administered instead of TAM. The patient complained of intense abdominal pain on the 35th day of administration. Laparotomy was then performed for her acute abdominal problem. Because necrosis from bleeding due to jejunal vein thrombosis was observed in the jejunum for about 15 cm, resection of the jejunum was carried out. Histological observation demonstrated thrombosis in the vein, and cellular infiltration around the thrombosis. The postoperative prognosis has been favorable and the postoperative course is now being monitored at our clinic (2 months after surgery). The patient has no complications such as diabetes mellitus or hypercholesterolemia. The thrombosis observed in the jejunal vein, which is a rare site for it on the 35th day of MPA administration was induced by MPA. Due attention must be paid to the formation of thrombosis when using MPA.


[Hydrea, an effective drug for chronic myelogenous leukemia]. Hydroxycarbamide (the brand name: Hydrea) was found effective to chronic myelogenous leukemia (CML) in Japan. In the preclinical study, this compound was active against mouse leukemia L 1210 and inhibited DNA synthesis. Clinically, Hydrea was given orally at the daily dose of 500-2,000 mg, dividing 1-3 times. For the maintenance therapy after remission induction, daily dose of 500-1,000 mg was given, dividing 1-2 times. As for the side effects, myelosuppression, disturbance of the gastro-intestinal tract and temporal liver and renal dysfunctions were observed. The response rate in the remission-induction therapy was as high as 92.1%.


Systemic steroids with or without 2% topical minoxidil in the treatment of alopecia areata. Thirty-two patients with mild to extensive alopecia areata, including 16 patients with alopecia totalis or universalis, entered a randomized, controlled trial of a 6-week taper of prednisone followed by either 2% topical minoxidil or vehicle applied three times daily for an additional 14 weeks. The results of this study were compared with an open trial of 48 patients with alopecia areata treated with a similar taper of prednisone with concomitant 2% topical minoxidil applied twice daily. Only terminal hair growth was considered and was quantitated as 1% to 24%, 25% to 49%, 50% to 74%, and 75% to 100%: only those with more than 25% terminal hair regrowth were considered to have had an objective response. At the end of 6 weeks of prednisone, 47% (15/32) of patients had more than 25% regrowth, including nine of 20 patients who had had at least 75% hair loss at baseline. Side effects of prednisone were primarily weight gain and mood changes/emotional lability. At 3 months, six of seven minoxidil-treated patients vs one of six vehicle-treated patients who had an objective response to prednisone maintained or augmented this hair growth: at the 20-week visit, these numbers were three of seven and zero of four patients, respectively. In the open trial, objective hair growth with prednisone was 30%, related to the extent of hair loss at baseline, and this growth persisted in more than 50% of patients at 6 months with the use of 2% topical minoxidil. A 6-week taper of prednisone offers potential for more than 25% regrowth in 30% to 47% of patients with alopecia areata with predictable and transient side effects. Two percent topical minoxidil three times daily appears to help limit poststeroid hair loss.


Serologic markers of gluten-sensitive enteropathy in bullous diseases. Dermatitis herpetiformis (DH) is characterized immunologically by the presence of IgA immune deposits in the skin and by the presence of various serum antibodies. Of these, antibodies to gliadin, reticulin, and endomysium have been found to be significant. There are, however, conflicting reports as to the exact specificity and sensitivity of these serologic markers in diagnosing DH. We examined the disease specificity of these three antibody markers in 14 patients with DH, in 98 patients with pemphigus and pemphigoid, and in 26 normal subjects. Reticulin and endomysium antibodies were detected by indirect immunofluorescence and gliadin antibodies by means of the enzyme-linked immunosorbent assay method. Among the various bullous diseases, endomysial and reticulin antibodies were found to be disease specific for DH. Endomysial antibodies occurred in twice the number of DH patients (72%) compared with the occurrence of reticulin antibodies (36%). Antigliadin antibodies were detected in two thirds of DH patients and were not disease specific since increased frequencies of these antibodies were also detected in patients with pemphigus and pemphigoid. These studies support the earlier findings of the high degree of specificity of endomysial antibodies for DH and, thus, help to differentiate DH from other bullous disorders.