Increased susceptibility of transfected prokaryotic and eukaryotic cells to antibiotic selection. At 39 to 40 degrees C, selection of antibiotic-resistant transfected mammalian cell lines or Escherichia coli required lower aminoglycoside antibiotic concentrations than at 37 degrees C. The thermosensitivity of antibiotic susceptibility was much more manifest during genetic selection experiments than in conventional growth inhibition assays.


In vitro activities of streptomycin and 11 oral antimicrobial agents against clinical isolates of Klebsiella rhinoscleromatis. We tested in vitro the activities of streptomycin and tetracycline--antibiotics that have long been used to treat rhinoscleroma--as well as several newer oral agents by using 23 isolates of the causative organism Klebsiella rhinoscleromatis. All isolates were inhibited by clinically achievable concentrations of trimethoprim-sulfamethoxazole, amoxicillin-clavulanate, chloramphenicol, ciprofloxacin, cephalexin, cefuroxime, and cefpodoxime.


In vitro susceptibilities of Borrelia burgdorferi to five oral cephalosporins and ceftriaxone. We determined the in vitro susceptibilities of eight Borrelia burgdorferi isolates to five oral cephalosporins. MICs for B. burgdorferi 297 were 23 micrograms/ml (cephalexin), 45 micrograms/ml (cefadroxil), 91 micrograms/ml (cefaclor), 0.13 microgram/ml (cefuroxime), 0.8 microgram/ml (cefixime), and 0.02 microgram/ml (ceftriaxone). When B. burgdorferi isolates were exposed to concentrations twice the MIC of cefuroxime, cefixime, or ceftriaxone, at least 72 h of incubation was required to kill 99% of the organisms.


Susceptibilities of ampicillin-resistant strains of Salmonella other than S. typhi to 10 antimicrobial agents. Ampicillin-resistant strains of Salmonella other than S. typhi constitute a health problem. We tested the antimicrobial susceptibilities to 10 antibiotics of 57 of these strains isolated in a 30-month period. The rates of resistance were as follows: chloramphenicol, 40.3%; tetracycline, 33.3%; gentamicin, 5.3%; co-trimoxazole, 5.3%; nalidixic acid, 1.8%; and amoxicillin-clavulanic acid, cefotaxime, ceftriaxone, aztreonam, and ciprofloxacin, 0%. In our experience, there are alternative antibiotics with excellent in vitro activities.


Standardized susceptibility testing of fluconazole: an international collaborative study. An international collaborative study of broth dilution (MIC) and disk diffusion susceptibility testing of fluconazole was conducted by using a chemically defined medium (High-Resolution Antifungal Assay Medium; Oxoid Ltd., Basingstoke, United Kingdom) and standard test methods performed in eight reference laboratories. Ten yeast isolates were tested by each test method in duplicate on each of 3 separate days. The intralaboratory reproducibility of the MIC test was excellent; 95.7% of the replicate tests (n = 220) were within 2 doubling dilutions of the other values in the set for the eight laboratories. The intralaboratory reproducibility of the disk test was also good, with 91% of the replicate tests (n = 234) agreeing with each other within an arbitrarily chosen value of 4 mm. Interlaboratory agreement of MIC test results was acceptable, with 84% of the MICs agreeing within 2 doubling dilutions. In contrast, the interlaboratory agreement of the disk test was not good, with only 59% of test results agreeing within 4 mm. Comparison of the rank order of MICs obtained in each laboratory with the reference rank order gave an agreement of 70 to 80% (median, 80%) with the MIC test and 70 to 90% (median, 80%) with the disk test. These preliminary results are encouraging for the development of standardized testing methods for testing fluconazole.


Prediction of human pharmacokinetics of panipenem-betamipron, a new carbapenem, from animal data. The pharmacokinetic behavior of panipenem (PAPM)-betamipron (BP), a new carbapenem, in humans was successfully predicted from data collected from six animal species. PAPM and BP were biphasically eliminated from plasma after intravenous (i.v.) administration of PAPM-BP to mice, guinea pigs, rats, rabbits, monkeys, and dogs. Elimination rates of PAPM and BP were correlated with animal size: the larger the animal was, the slower the elimination was. As for PAPM and BP, log-log plots of total plasma clearance (CLtot) versus body weight and log-log plots of distribution volume at steady state (VSS) versus body weight for six animal species were linear, with high correlation coefficients. These allometric equations were extrapolated to predict CLtot and VSS for PAPM and BP in humans. In addition, concentration in plasma-time profiles for humans were predicted by using two-exponent equations fitted to the complex Dedrick plot of animal data. Predicted values for CLtot and VSS for PAPM and BP in humans agreed well with observed values in humans given 750/750 mg of PAPM-BP as an i.v. drip infusion for 30 min. Predicted concentration in plasma-time profiles for humans approximated observed profiles. Thus, the pharmacokinetics of PAPM-BP extrapolated well from animal species to humans when allometric equations and the complex Dedrick plot were used.


Nucleotide sequences of CAZ-2, CAZ-6, and CAZ-7 beta-lactamase genes. CAZ-2, CAZ-6, and CAZ-7 are plasmid-mediated beta-lactamases that are markedly active against ceftazidime. The corresponding structural genes were amplified by the polymerase chain reaction. Nucleotide sequences were determined by direct sequencing of the amplified products. Analysis of the nucleotide and the deduced amino acid sequences showed that CAZ-2, CAZ-6, and CAZ-7 are derived from TEM-2 by three, four, and two amino acid substitutions, respectively. All these substitutions are located at positions 102, 162, 235, 236, and 237 (Sutcliffe numbering), which are known to extend the substrate range of beta-lactamases. These substitutions are Lys-102, Ser-162, and Ser-236 in CAZ-2; Lys-102, Ser-162, Thr-235, and Lys-237 in CAZ-6; and Lys-102 and His-162 in CAZ-7. These results indicate that the nucleotide sequence of CAZ-2 is identical to that of TEM-8. The nucleotide sequence of CAZ-7 possesses the two mutations described in TEM-16 by the oligotyping method. In contrast, the combination of mutations encountered in CAZ-6 has not yet been described, and this enzyme was designated TEM-24.


Primary and secondary metabolism of pentamidine by rats. The antiprotozoal drug pentamidine [1,5-bis(4'-amidinophenoxy)pentane] has been previously shown to be metabolized by rat liver microsomes, and five of the seven putative primary metabolites have been identified. With the synthesis and identification of 5-(4'-amidinophenoxy)pentanoic acid and 5-(4'-amidinophenoxy)-1-pentanol as the remaining two metabolites, the primary metabolism of pentamidine in rats appears fully characterized. Use of [14C]pentamidine with rat liver microsomes confirms this conclusion, since no unidentified radioactive peaks were detected by high-performance liquid chromatography (HPLC). Isolated, perfused rat livers were used with [14C]pentamidine to identify secondary metabolites. Only two novel radioactive peaks were detected by HPLC analysis of perfused liver samples. The treatment of liver samples with sulfatase or beta-glucuronidase resulted in the reduction or elimination of these peaks and gave rise to peaks identified as para-hydroxybenzamidine and 5-(4'-amidinophenoxy)pentanoic acid. It was concluded from these results that only these two primary metabolites were conjugated with sulfate or glucuronic acid. After 4 h of incubation in the perfused liver system, approximately 15% of the recovered radiolabel was pentamidine. These results suggest that pentamidine metabolism can be rapid and extensive in rats.


Effect of concomitant administration of piperacillin on the dispositions of isepamicin and gentamicin in patients with end-stage renal disease. Piperacillin inactivation of the aminoglycosides isepamicin and gentamicin in 12 chronic hemodialysis patients was assessed. Six subjects each received isepamicin (7.5 mg/kg of body weight) or gentamicin (2 mg/kg) alone and in combination with piperacillin (4 g every 12 h for four doses). Isepamicin and gentamicin concentrations in plasma and urine were monitored over 48 h after each dose and analyzed by high-performance liquid chromatography and fluorescence polarization immunoassay, respectively. The pharmacokinetics of isepamicin were not significantly altered during combination treatment with piperacillin. The total body clearance (3.79 +/- 0.71 versus 3.94 +/- 1.05 ml/min), the steady-state volume of distribution (0.19 +/- 0.04 versus 0.18 +/- 0.03 liter/kg), and the terminal elimination half-life (47.91 +/- 7.20 versus 45.08 +/- 10.34 h) were not significantly altered in the presence of piperacillin. In contrast, the terminal elimination half-life (47.68 +/- 20.58 versus 35.67 +/- 11.18 h) of gentamicin was significantly reduced when gentamicin was given with piperacillin. The total body clearance (4.26 +/- 3.07 versus 4.89 +/- 1.94 ml/min) and the steady-state volume of distribution (0.19 +/- 0.04 versus 0.20 +/- 0.04 liter/kg) of gentamicin were not significantly altered during combination therapy; however, the nonrenal clearance of gentamicin administered in combination with piperacillin (3.56 +/- 0.38 ml/min) increased significantly compared with that of gentamicin (2.03 +/- 0.50 ml/min) given alone. The results of this study suggest that no additional dosage adjustment of isepamicin during concomitant therapy with piperacillin in hemodialysis patients is necessary. However, this does not preclude the need for appropriately ex vivo-handled specimens for monitoring isepamicin concentrations in plasma to ensure therapeutic efficacy and prevent toxicity. Furthermore, additional dosage adjustments may be necessary when gentamicin is used concomitantly with piperacillin, on the basis of the significant in vivo inactivation that takes place in end-stage renal disease patients.


Cross-resistance to meropenem, cephems, and quinolones in Pseudomonas aeruginosa. Multiple-drug-resistant mutants were isolated from Pseudomonas aeruginosa PAO1 on agar plates containing ofloxacin and cefsulodin. These mutants were four to eight times more resistant to meropenem, cephems, carbenicillin, quinolones, tetracycline, and chloramphenicol than the parent strain was. In contrast, these mutants showed no significant changes in their susceptibilities to all carbapenems except meropenem. In these mutants, the amounts of an outer membrane protein with an apparent molecular weight of 49,000 (designated OprM) were increased compared with the amount in PAO1. Multiple-drug-resistant mutants of this type were also isolated from PAO1 on agar plates containing meropenem. Approximately 5% of clinical isolates showed cross-resistance to meropenem, cephems, and quinolones, concomitant with overproduction of OprM. Moreover, these two phenotypes, i.e., multiple-drug resistance and overproduction of OprM, were cotransferable by transduction. These data suggest that overproduction of OprM is associated with cross-resistance to meropenem, cephems, and quinolones in P. aeruginosa. The ofloxacin-cefsulodin-resistant mutant required higher concentrations of meropenem to induce beta-lactamase than PAO1 did, indicating the possibility that this mutation involves decreased outer membrane permeability to meropenem.


Liquid chromatographic determination of amoxicillin concentrations in bovine plasma by using a tandem solid-phase extraction method. We report a means of determining amoxicillin in bovine plasma by liquid chromatography with UV detection at 235 nm. Purification and concentration of extracts were accomplished by a tandem solid-phase extraction procedure with two reversed-phase columns. Separation of amoxicillin from interferences was improved by the incorporation of a crown ether in the solvent systems used both for the solid-phase extraction and the final high-pressure liquid chromatography. Cefadroxil was added as an internal standard. The average recovery of amoxicillin from plasma (n = 23) was 78.2 +/- 3.0%, and the within-run and between-run coefficients of variation ranged from 1.8 to 7.0%. The detection limit was estimated at 0.1 microgram/ml. This method was used to determine amoxicillin in bovine plasma after intramuscular administration of the drug.


Detection of extended-spectrum beta-lactamases in members of the family Enterobacteriaceae: comparison of the double-disk and three-dimensional tests. The three-dimensional and clavulanate double-disk potentiation tests were compared as procedures for the detection of extended-spectrum beta-lactamase production in 32 strains of Escherichia coli and Klebsiella pneumoniae, 31 of which produced TEM-1, TEM-2, TEM-3, TEM-4, TEM-5, TEM-7, TEM-8, TEM-9, TEM-10, TEM-12, TEM-101, SHV-1, SHV-2, SHV-3, SHV-4, SHV-5, CAZ-2, MIR-1, or an unidentified extended-spectrum beta-lactamase with a pI of 5.95, with some strains producing multiple beta-lactamases. The three-dimensional test, which was performed in conjunction with a routine disk diffusion test, detected extended-spectrum beta-lactamase production in 26 of 28 (93%) of the strains that produced extended-spectrum beta-lactamases. The clavulanate double-disk potentiation test detected extended-spectrum beta-lactamases in only 22 of the 28 strains (79%) when it was performed as currently recommended. The three-dimensional test, when performed in conjunction with the disk diffusion test, offered the advantages of providing simultaneous information about both antibiotic susceptibility and extended-spectrum beta-lactamase production, coupled with a greater sensitivity and earlier detection of extended-spectrum beta-lactamases.


In vitro and in vivo antibacterial activities of E1077, a novel parenteral cephalosporin with a broad antibacterial spectrum. E1077 is a new injectable cephalosporin with a broad spectrum of antibacterial activity against gram-positive and gram-negative bacteria, including staphylococci and Pseudomonas aeruginosa. The in vitro activities of E1077 against clinical isolates of methicillin-susceptible Staphylococcus aureus (MIC of E1077 for 90% of the strains tested [MIC90], 0.78 microgram/ml) and methicillin-resistant S. aureus (MIC90, 50 micrograms/ml) were similar to those of cefpirome and flomoxef. Against Enterococcus faecalis (MIC90, 6.25 micrograms/ml), E1077 was the most active of the drugs tested and four times more active than cefpirome. The MIC90S of E1077 for streptococci, Haemophilus influenzae, and Neisseria gonorrhoeae ranged from 0.05 to 0.78 microgram/ml; E1077 was similar in activity to cefpirome. E1077 inhibited 90% of most species of the family Enterobacteriaceae at concentrations of less than or equal to 1.56 micrograms/ml, with the exception of Serratia marcescens and Proteus vulgaris (12.5 micrograms/ml). The activity of E1077 against P. aeruginosa (MIC90, 6.25 micrograms/ml) was comparable to that of ceftazidime. In vivo activity was evaluated with systemic infections in mice. E1077 showed a protective effect against systemic infections by gram-positive or gram-negative bacteria, as reflected by its in vitro activity. The protective effects of E1077 were higher than those of cefpirome against S. aureus and P. aeruginosa infections and similar to those of cefpirome against other bacterial infections. Morphological studies using differential interference and phase-contrast microscopy showed that low concentrations of E1077 caused swelling of S. aureus and spheroplast and bulge formation in P. aeruginosa. In general, the antibacterial profile of E1077 is similar to that of cefpirome.


Diffusion of meropenem and imipenem through the outer membrane of Escherichia coli K-12 and correlation with their antibacterial activities. The outer membrane permeability to meropenem and imipenem in Escherichia coli K-12 was investigated, and its porin-deficient mutants were transformed with a constructed vector carrying the carbapenem-hydrolyzing CphA metallo-beta-lactamase gene. By using the method of Zimmermann and Rosselet, meropenem was shown to penetrate through the outer membrane of E. coli K-12 five times faster than cephaloridine but twice as slowly as imipenem. Lack of one or both porins significantly reduced the penetration of both carbapenems. No evidence of specific porin pathways of the type described in Pseudomonas aeruginosa was found. Despite its slower penetration, meropenem was two to eight times more active than imipenem against both parent and porin-defective mutants, whether harbouring CphA beta-lactamase or not. Meropenem was also more active than imipenem against E. coli DC2, a strain with a breakdown in the outer membrane permeability which made periplasmic concentrations of beta-lactams similar to the external concentrations. In this strain, meropenem caused a more than 50% reduction in cell number increase at a concentration very close to the 50% inhibitory concentration for penicillin-binding protein type 2 (PBP 2), whereas imipenem, at the same concentration, did not significantly inhibit cell growth. This result was explained by the higher affinity of meropenem for PBP 3 compared with imipenem and supports the conclusion that synergistic inhibition of both PBPs was the main mechanism in the better antibacterial activity of meropenem.


Chitin biosynthesis in Candida albicans grown in vitro and in vivo and its inhibition by nikkomycin Z. An N-acetyl-D-[14C]glucosamine radiolabel incorporation assay has been used to monitor chitin biosynthesis in whole cells of Candida albicans both in vitro and in vivo in two different mouse infection models, one using the peritoneal cavity as a chamber in which to add and retrieve cells and the other using infected kidneys. Specific labeling of chitin in alkali-insoluble material was confirmed by chitinase digestion, analysis of acid hydrolysates, and the use of nikkomycin Z as a probe. Nikkomycin Z was shown to strongly inhibit chitin biosynthesis in C. albicans grown in vitro and in vivo in both models. This demonstrates that nikkomycin Z-susceptible chitin synthase activity is present in C. albicans when the fungus is in its pathogenic state in vivo. The limited use of nikkomycin as a therapeutic agent is discussed.


Potential effects of erythromycin on host defense systems and virulence of Pseudomonas aeruginosa. We evaluated several potential effects of erythromycin (EM) on host defense systems and the virulence of Pseudomonas aeruginosa. Peritoneal macrophages obtained from mice given 250 mg of EM per kg of body weight for 7 days by the intraperitoneal, intravenous, subcutaneous, or oral route produced significantly greater amounts of thymocyte-activating factors. These data suggest that EM enhances the in vivo production of cytokines, such as interleukins 1 and 6. Treatment of P. aeruginosa D4 with subinhibitory concentrations of EM enhanced the association of bacteria with murine Kupffer cells in vitro and increased bacterial clearance from the blood in mice. EM suppressed the in vitro production of exotoxin A, total protease, elastase, and phospholipase C by P. aeruginosa D4; exotoxin A production by P. aeruginosa PA-103; and total protease production by P. aeruginosa B16 and PAO1 in a generally dose-dependent manner. These data demonstrate that EM produces various effects in addition to its direct antimicrobial activity, suggesting that it has potential as an immunomodulator or bacterial virulence-suppressing agent against P. aeruginosa and other infections.


In vitro activity of L-627, a new carbapenem. The in vitro activity of L-627, a new parenterally administered carbapenem, was compared with those of imipenem, meropenem, FCE 22101 (a penem), ceftazidime, and ceftriaxone. L-627 was active against members of the family Enterobacteriaceae (MIC for 90% of strains tested [MIC90] ranging from 0.03 to 4 micrograms/ml). L-627 displayed activity equal to that of meropenem against Pseudomonas aeruginosa (MIC90, 2 micrograms/ml), although, as with other carbapenems, the antipseudomonal activity was reduced against D2-deficient strains. Staphylococci and streptococci were susceptible (MIC90 of 1.0 micrograms/ml for Staphylococcus aureus and 0.015 micrograms/ml for group A streptococci). L-627 also had activity against anaerobic bacteria (MIC90, 2.0 micrograms/ml for Bacteroides fragilis). Neisseria gonorrhoeae and Neisseria meningitidis were highly susceptible (MIC90, 0.06 micrograms/ml), and against the common respiratory pathogens (Haemophilus influenzae, Streptococcus pneumoniae, and Moraxella catarrhalis), the MIC90s were less than or equal to 2.0 micrograms/ml. The protein binding of L-627 ranged from 13.8 to 22%, depending on the concentration. The presence of human serum had little effect on the MIC or MBC of L-627. These results suggest that L-627 merits further study in the treatment of infections caused by a wide range of pathogens.


Activities of antifolate, antiviral, and other drugs in an immunosuppressed rat model of Pneumocystis carinii pneumonia. The efficacy of antifolate, antiviral, and other drugs was compared in an experimental model of pneumocystosis. Sulfamethoxazole (SMX) administered alone in doses of greater than or equal to 60 mg/kg/day was highly effective in treatment and prophylaxis. Low (less than or equal to 15 mg/kg/day) doses of SMX showed limited, dose-related anti-Pneumocystis carinii activity in therapy but were more effective in prophylaxis. The dihydrofolate reductase (DHFR) inhibitors trimethoprim (TMP), pyrimethamine, and trimetrexate exhibited little anti-P. carinii activity when administered alone and did not enhance the efficacy of SMX; the effects of the DHFR inhibitors could not be related to the dose or the concentration in serum. These data suggested that the rat model is an excellent system for studying the anti-P. carinii activity of sulfonamides but is of limited value in studying DHFR inhibitors. The antiviral drugs azidothymidine, dideoxyinosine, inosine pranobex (Isoprinosine), amantadine, and acyclovir displayed little or no activity against P. carinii; however, azidothymidine did not impair the efficacy of SMX or TMP-SMX. These results supported the clinical practice of giving antiviral agents together with antifolate drugs to patients infected with human immunodeficiency virus and suggested that the beneficial effects of antiviral agents on the occurrence of pneumocystosis are due mainly to their effects on the virus or the host immune response. In contrast to the antiviral drugs, 9-deazainosine, a nucleoside analog with antiprotozoal properties, demonstrated marked activity against P. carinii which was related to dose and route of administration. These data raised the possibility that anti-P. carinii activity is a general property of purine nucleosides and suggested that further exploration of this class of compounds might lead to clinically useful agents.


Treatment of experimental pneumocystosis: review of 7 years of experience and development of a new system for classifying antimicrobial drugs. Over a 7-year period, we analyzed 261 dose regimens of antimicrobial drugs in the treatment and prevention of Pneumocystis carinii pneumonia in an immunosuppressed rat model. These compounds ranged from drugs in clinical use to newly synthesized agents. Drug efficacy was expressed as the magnitude of the reduction in median P. carinii cyst or nucleus counts on a scale ranging from inactive (less than 5-fold) to very markedly active (greater than or equal to 1,000-fold). The classification system was reproducible and allowed drugs studied at different times to be compared with each other. The system demonstrated a hierarchy of anti-P. carinii activity not only among classes of compounds but also among individual members of a drug class. Sulfonamides, sulfones, and diamidines were the most active agents; some purine nucleosides and nitrofurans also showed promising activity; and most antiparasitic, antifungal, antibacterial, and antiviral drugs were inactive. We conclude that this classification system represents a simple, quantitative method of comparing the activities of antimicrobial drugs against P. carinii. Information gained from this system should be helpful in developing new anti-P. carinii compounds and establishing standard procedures for their evaluation.


Adaptive resistance following single doses of gentamicin in a dynamic in vitro model. Adaptive resistance is a phenomenon recently described for Pseudomonas aeruginosa and other gram-negative bacilli following exposure to aminoglycoside antibiotics. It is a reversible form of resistance which develops within 1 to 2 h of initial exposure to an aminoglycoside and disappears several hours after removal of the antibiotic. We investigated adaptive resistance in P. aeruginosa ATCC 27853 following single doses of gentamicin by using a dynamic in vitro model which mimics in vivo pharmacokinetics. The initial peak gentamicin concentrations were 2.5, 8, and 25 mg/liter, and these were followed by an exponential decay in the concentration, with a half-life of 2.5 h. The degree of adaptive resistance was greater and the duration was longer with higher initial gentamicin concentrations. Maximal adaptive resistance occurred between 2 and 10 h following 8 mg/liter and between 2 and 16 h following 25 mg/liter. Full recovery of susceptibility occurred at approximately 36, 39, and 43 h following 2.5, 8, and 25 mg/liter, respectively, at which times the gentamicin concentrations were extremely low. Longer dosing intervals for aminoglycosides may improve efficacy by allowing time for adaptive resistance to resolve.


Pharmacokinetics of [14C]FCE 22891, a penem antibiotic, following oral administration to healthy volunteers. FCE 22891 is a prodrug of the penem antibiotic FCE 22101 and is suitable for oral administration. The pharmacokinetics of FCE 22891 were investigated in four healthy male volunteers following the oral administration of 500 mg of [14C]FCE 22891. Levels of radioactivity in plasma were always higher and persisted for longer than those of FCE 22101. The time to the maximum concentration of radioactivity in plasma generally coincided with that of FCE 22101. The respective values for the maximum concentrations of radioactivity in plasma were, on average, 8.57 +/- 2.95 micrograms equivalent/ml and 2.97 +/- 2.05 micrograms/ml. Over a 5-day period, mean urinary and fecal recovery of radioactivity accounted for 53.2 and 41.0% of the dose, respectively. The average amount of FCE 22101 excreted in urine and feces corresponded to 9.0 and 1.6% of the dose, respectively. The urinary recovery of the open-ring metabolite P1 and of its 5-S epimer P2 accounted for about 6.5 and 1.2% of the dose, respectively. Other chromatographic peaks corresponding to nonidentified compounds accounted for about 14.0% (polar metabolite fraction; peak P), 3.7% (less polar fraction; peak X), and 15.4% (least polar fraction) of the dose. Elimination of radioactivity and FCE 22101 in urine was rapid. Intersubject variability in the kinetics of total radioactivity in plasma was far less than that observed for FCE 22101. The results of the present study support suggestions that presystemic metabolism of FCE 22101 and/or transformation of the prodrug to compounds other than FCE 22101 are the main cause of intersubject variability in the kinetics of FCE 22101 produced in plasma following oral administration of its prodrug.


Antipneumocystis activity of water-soluble lipopeptide L-693,989 in rats. Water-soluble lipopeptide L-693,989 was evaluated for its antipneumocystis activity in rats. Rats from colonies with latent Pneumocystis carinii infections were immunosuppressed with dexamethasone for 6 weeks to facilitate the development of acute P. carinii pneumonia (PCP). After 6 weeks, the rats were maintained on dexamethasone and were treated twice daily for 4 days with various concentrations of L-693,989. At a dose of 0.15 mg/kg of body weight, the compound effectively eliminated 90% of the cysts in 4 days. Trophozoite forms of P. carinii were still present in these animals, as determined by using a P. carinii-specific DNA probe. A 3-week therapy study showed that the trophozoite load did not expand during treatment and that the trophozoites already present at the initiation of therapy appeared to persist. This may be a consequence of the stage specificity of the compound for cyst development and the severe immunosuppressive effects of dexamethasone on rats. When evaluated as a daily parenteral prophylactic agent, L-693,989 was effective in preventing the development of both P. carinii cysts and trophozoites, demonstrating its potential for use in prophylaxis and implying that the cyst stage of P. carinii is an obligatory step in trophozoite multiplication. The foamy exudate commonly associated with P. carinii infections was absent in the lungs of rats on prophylaxis. The compound was also evaluated via oral administration and was found to have a 90% effective dose of 32 mg/kg for therapy of acute infections and 5 mg/kg for daily prophylaxis.


Studies of the effect of a platelet-activating factor antagonist, CL 184,005, in animal models of gram-negative bacterial sepsis. The effect of CL 184,005, a potent and specific platelet-activating factor antagonist, has been examined in a variety of animal models relevant to gram-negative bacterial sepsis. Pretreatment of mice with CL 184,005 protected them from the lethal effects of platelet-activating factor. When rats or primates rendered hypotensive with endotoxin were treated with CL 184,005, blood pressure was normalized. Pretreatment of rats with CL 184,005 protected them from the gastrointestinal lesions induced by endotoxin. Pretreatment of rats and mice with CL 184,005 protected them from the lethal effects of endotoxin. Plasma tumor necrosis factor levels in endotoxin-treated mice were lower when the mice were pretreated with CL 184,005. These observations suggest that CL 184,005 may be potentially useful in the treatment of gram-negative bacterial sepsis, and the agent is undergoing clinical evaluation.


Activity of amphotericin B cholesterol dispersion (Amphocil) in experimental visceral leishmaniasis. Standard therapy of human visceral leishmaniasis with parenteral pentavalent antimonial agents is generally curative but has the disadvantages of a 28-day treatment course, occasional treatment failures, and toxicity. The antifungal and antileishmanial agent amphotericin B has been complexed with lipids to develop a less toxic formulation of amphotericin B. Because lipid particles are phagocytized by the reticuloendothelial system, lipid-associated amphotericin B should be concentrated in infected macrophages and be very effective against visceral leishmaniasis. One formulation, amphotericin B cholesterol dispersion (ABCD) (Amphocil), was tested for antileishmanial activity in Leishmania donovani-infected hamsters. In the first experiment, hamsters were infected, administered with the drug 3 days later, and then sacrificed after a further 4 days. ABCD (dose needed to suppress 99% of hepatic parasites compared with controls [SD (99)], 0.4 mg/kg of body weight) was 15 times as effective as conventional amphotericin B [SD (99), 6.0 mg/kg]. Pentavalent antimony in the form of meglumine antimonate had an SD (84) of 416 mg/kg. In a second experiment in which animals were allowed to become more heavily infected, the drug was administered 10 days after infection and the animals were sacrificed after a further 2, 7, or 11 days. ABCD was approximately four times as active as conventional amphotericin B. These experiments suggest that ABCD is at least four times as active as conventional amphotericin B against visceral leishmaniasis and that clinical trials are warranted.


Activities of clarithromycin against eight slowly growing species of nontuberculous mycobacteria, determined by using a broth microdilution MIC system. MICs of clarithromycin against 324 clinical isolates belonging to eight species of slowly growing nontuberculous mycobacteria were determined by using a broth microdilution system. Isolates were inoculated into twofold drug dilutions in Middlebrook 7H9 broth (pH corrected to 7.4) and then incubated at 30 degrees C for 7 days for Mycobacterium marinum and for 14 days for all other species. The MIC for 90% of the strains (MIC90) was less than or equal to 0.5 micrograms/ml for isolates of Mycobacterium gordonae (6 strains), Mycobacterium scrofulaceum (5 strains), Mycobacterium szulgai (6 strains), and Mycobacterium kansasii (35 strains). MICs for M. marinum (25 strains) and Mycobacterium avium complex (237 strains) were higher, but 100% and 89% of the strains, respectively, were susceptible to less than or equal to 4 micrograms/ml. In contrast, MICs for five of six M. simiae strains were greater than 8 micrograms/ml, and the range of MICs for Mycobacterium nonchromogenicum varied from less than or equal to 0.125 to 8 micrograms/ml. For the 237 isolates of M. avium complex, the MIC50 was 2 micrograms/ml and the MIC90 was 8 micrograms/ml. MICs for most isolates (77%) were in the 1- to 4-micrograms/ml range. For the 80 isolates in this group known to be from AIDS patients, the MIC50 was 4 micrograms/ml and the MIC90 was 8 micrograms/ml. These MIC studies combined with preliminary clinical trials suggest that clarithromycin may be useful for drug therapy of most species of the slowly growing nontuberculous mycobacteria except M. simiae.


Outbreak of ceftazidime resistance due to a novel extended-spectrum beta-lactamase in isolates from cancer patients. Ceftazidime is widely used in the therapy of infectious complications in neutropenic patients. We studied an outbreak of ceftazidime-resistant gram-negative bacillary infections in pediatric cancer patients receiving empirical ceftazidime therapy for neutropenic fever. Fourteen isolates (12 Klebsiella pneumoniae and 2 Escherichia coli) from 13 patients were studied. Specimens were obtained from multiple clinical sites including blood, urine, throat, and lung. The organisms were resistant to ceftazidime, aztreonam, and penicillins but remained susceptible to cephamycins and imipenem. All resistant isolates produced a novel beta-lactamase (TEM-26) with a pI of approximately 5.58, which was transferred by transformation to E. coli on a 7.9-kb nonconjugative plasmid which cotransferred resistance to trimethoprim-sulfamethoxazole. This enzyme readily hydrolyzed ceftazidime, aztreonam, and penicillins in a spectrophotometric assay. DNA sequencing data suggest that TEM-26 is derived from TEM-1.


Vancomycin resistance gene vanC is specific to Enterococcus gallinarum. Nearly all strains of Enterococcus gallinarum are resistant to low levels of vancomycin. The glycopeptide resistance gene vanC from E. gallinarum BM4174 has recently been cloned and sequenced. A probe specific for vanC hybridized with a 2.7-kb EcoRI and a 4.5-kb HindIII fragment of total DNA from the 42 strains of E. gallinarum studied. No homology was detected with DNA of strains belonging to other species intrinsically resistant to vancomycin, including Enterococcus casseliflavus, a species that expresses a vancomycin resistance phenotype similar to that of E. gallinarum. No hybridization with DNA of enterococcal strains with acquired resistance to high or low levels of vancomycin was observed. The specificity of the vanC probe allowed us to distinguish E. gallinarum from 12 other species of enterococci, indicating that this probe is a useful tool for species identification within the genus Enterococcus.


Presence of Clostridium difficile and antibiotic and beta-lactamase activities in feces of volunteers treated with oral cefixime, oral cefpodoxime proxetil, or placebo. Three groups of six healthy adult volunteers were randomly assigned to a treatment with 400 mg of oral cefpodoxime proxetil, oral cefixime, or placebo per day for 10 days. Informed consent was obtained from all volunteers. Clostridium difficile was not detected in the feces of any subject before treatment or at any time in the subjects in the placebo group. C. difficile was, however, detected in all subjects treated with cefpodoxime proxetil and in five of six treated with cefixime. Genomic DNA restriction patterns showed that the strains of C. difficile differed from one volunteer to another. Two subjects both shed different strains at different times during the 25-day surveillance period. All isolates were resistant to cefixime and cefpodoxime (MIC for 90% of strains, 256 and 512 mg/liter, respectively). Antibiotic activity was found in the feces of one volunteer treated with cefpodoxime proxetil and of four volunteers treated with cefixime. It was inversely correlated with the presence of fecal beta-lactamase activity. Intestinal side effects were limited to modifications of stool consistency, which occurred in only 3 of the 12 treated volunteers and did not lead to cessation of treatment. These modifications were significantly associated with the presence of fecal antibiotic activity (P less than 0.05) but not with the shedding of toxigenic or nontoxigenic strains of C. difficile or with the presence of toxin A in feces, which was detected only in one perfectly healthy treated volunteer.


Antimicrobial prophylaxis for major head and neck surgery in cancer patients: sulbactam-ampicillin versus clindamycin-amikacin. A total of 99 patients with head and neck cancer who were to undergo surgery were randomized in a prospective comparative study of sulbactam-ampicillin (1:2 ratio; four doses of 3 g of ampicillin and 1.5 g of sulbactam intravenously [i.v.] every 6 h) versus clindamycin (four doses of 600 mg i.v. every 6 h)-amikacin (two doses of 500 mg i.v. every 12 h) as prophylaxis starting at the induction of anesthesia. The two groups of evaluable patients (43 in the clindamycin-amikacin treatment group and 42 in the sulbactam-ampicillin treatment group) were comparable as far as age (mean, 57 years; range, 21 to 84 years), sex ratio (71 males, 28 females), weight (mean, 66 kg; range, 40 to 69 kg), indication for surgery (first surgery, 48 patients; recurrence, 37 patients), previous anticancer treatment (surgery, radiation therapy, chemotherapy), type of surgery, and stage of cancer. The overall infection rate (wound, bacteremia, and bronchopneumonia) within 20 days after surgery was 20 patients in each group. Wound infections occurred in 14 (33%) sulbactam-ampicillin-treated patients and 9 (21%) clindamycin-amikacin-treated patients (P = 0.19; not significant). The rates of bacteremia were 2 and 4%, respectively. The rates of bronchopneumonia were 14.3 and 23.2%, respectively (P was not significant). Most infections were polymicrobial, but strict anaerobes were recovered only from patients who received sulbactam-ampicillin. Antimicrobial treatment was required within 20 days after surgery for 42% of the sulbactam-ampicillin-treated patients and 44% of the clindamycin-amikacin-treated patients. By comparison with previous studies, we observed a decreased efficacy of antimicrobial prophylaxis in patients with head and neck cancer undergoing surgery because of the increased proportion of patients who were at very high risk for infection (extensive excision and plastic reconstruction in patients with recurrent stage III and IV cancers) and because of the longer duration of surgery.


Greatly enhanced inhibition of human immunodeficiency virus type 1 replication in CEM and HT4-6C cells by 3'-deoxythymidine diphosphate dimyristoylglycerol, a lipid prodrug of 3'-deoxythymidine. 3'-Deoxythymidine (3dT) is a weakly active dideoxynucleoside in human immunodeficiency virus (HIV)-infected cells because of its slow phosphorylation by cellular thymidine kinase. 3dT diphosphate dimyristoylglycerol (3dTDP-DMG), a phospholipid prodrug, was synthesized and found in vitro to be 18- to 50-fold more effective than 3dT in CEM and HT4-6C cells. In CEM cells, the selectivity index of 3dTDP-DMG was 270 versus 48 for 3dT, an increase of 5.6-fold. In thymidine kinase-deficient mutant CEM cells infected with HIV, 3dT and zidovudine (AZT) were virtually inactive but 3dTDP-DMG retained substantial activity, suggesting that its greatly increased antiviral activity is due in part to bypass of thymidine kinase. 3dTDP-DMG was 14- to 37-fold more active than 3dT in AZT-sensitive and AZT-resistant clinical isolates of HIV; no cross-resistance with AZT was noted. The results suggest that lipid prodrugs may be utilized in some cases to confer unique metabolic advantages over the corresponding free nucleoside; in the case of 3dTDP-DMG, an 18- to 50-fold increase in antiretroviral activity was observed in LAV-infected cells. The strategy would seem to be especially useful for antiviral nucleosides which are poorly phosphorylated.


In vitro susceptibility of Mycobacterium kansasii to clarithromycin. The MICs of the macrolide clarithromycin for 31 clinical isolates of Mycobacterium kansasii were determined by three different methods. The methods employed were the proportion resistance method on 7H10 agar, the radiometric (BACTEC) method, and the T100 method of datum analysis. All methods gave similar results. The MICs were in a narrow range from 0.16 to 0.50 microgram/ml, with the MICs for 90% of isolates tested of 0.50 microgram/ml for the agar dilution and radiometric methods and 0.37 microgram/ml for the T100 method. The MBCs were determined for nine representative isolates by the radiometric broth method. The MBCs were equal to the MICs for four isolates, and the MBCs were twofold higher than the MICs for five isolates. Killing of 99.9% of the bacterial population was achieved at a clarithromycin concentration of 2.0 micrograms/ml for all nine isolates tested.


Inhibition of intrahepatic hepatitis B virus replication by antiviral drugs in a novel transgenic mouse model. Transgenic mice which carry the integrated human hepatitis B virus (HBV) genome and produce HBV particles in the liver were treated with antiviral compounds, and the amount of viral DNA in the liver and serum was monitored by Southern analyses. Mouse alpha interferon (200,000 U per injection, twice daily for 3 days) and a novel nucleoside, oxetanocin G (15 mg/kg of body weight per injection, twice daily for 7 days), inhibited viral DNA synthesis in the liver almost completely. This animal model should prove useful in evaluating anti-HBV agents.


Susceptibilities of members of the Bacteroides fragilis group to 11 antimicrobial agents. The susceptibilities of 200 clinical isolates of the Bacteroides fragilis group to 11 antimicrobial agents were determined by the broth microdilution method of the National Committee for Clinical Laboratory Standards. All isolates were susceptible to imipenem and ticarcillin-clavulanic acid. The rates of resistance to cefoxitin and clindamycin were low (4 and 6%, respectively), while those to ceftizoxime and cefotetan were higher (10.5 and 24%, respectively).


Pseudomonas aeruginosa biofilm as a diffusion barrier to piperacillin. Pseudomonas aeruginosa 579 biofilms formed on dialysis membranes retarded piperacillin diffusion. Treatment of biofilms with 5.0 mM CaCl2.2H2O prevented diffusion. Biofilms permitted equilibration of [14C]glucose. Thin-layer chromatography of fluids distal to untreated (viable and nonviable) and viable Ca(2+)-treated P. aeruginosa 579 biofilms and fluids distal to a viable P. aeruginosa mutant noninducible for the expression of beta-lactamase did not detect piperacillinoic acid.


Effect of biliary obstruction on the hepatic excretion of imipenem-cilastatin. The biliary excretion of imipenem-cilastatin studied by endoscopic cannulation of the common bile duct in patients with complete obstruction and in a group without obstruction showed that despite a 24-h prophylaxis, the bile obtained from patients with obstruction immediately after cannulation contained neither imipenem nor cilastatin, while there were 2 and 5% of peak concentrations in serum for imipenem and cilastatin, respectively, in the bile from patients without obstruction. Biliary excretion of both compounds increased rapidly after decompression, reaching a maximum of 15% of peak levels in serum within 2 h. Twenty-four hours after drainage, the biliary excretion of the drugs further improved. We conclude that since biliary obstruction impairs excretion of antibiotics, drainage is necessary for the control of sepsis in obstructed cholangitis.


Immunotoxic potential of antiviral drugs: effects of ganciclovir and (S)-1-(3-hydroxy-2-phosphonylmethoxy propyl) cytosine on lymphocyte transformation and delayed-type hypersensitivity responses. In the present studies, we examined the in vitro and in vivo effects of ganciclovir (DHPG) and a relatively new nucleoside analogue, (S)-1-(3-hydroxy-2-phosphonylmethoxy propyl) cytosine (HPMPC), on lymphocyte responses to T cell mitogens and delayed-type hypersensitivity (DTH) responses to dinitrofluorobenzene (DNFB). Initially, responses of mouse splenic mononuclear cells and human peripheral blood mononuclear cells to PHA and con A were evaluated in vitro in the presence of each drug. Both drugs inhibited the responses to each mitogens; however, DHPG had a greater inhibitory effect on con A responses of human and mouse lymphocytes than did HPMPC. Also, spleen cells from mice treated for 7 days with DHPG responded less well to PHA stimulation than cells from untreated or HPMPC-treated mice. No effect of either drug was observed on con A responses. Treatment of mice with either drug decreased the development of DTH responses, with HPMPC having a greater inhibitory effect than DHPG. The results from the present studies suggest that both DHPG and HPMPC may have inhibitory effects on the development of certain immune functions at high dosages, but at drug concentrations that were therapeutic in animal model studies, little inhibitory effects were observed.


Inhibition of human immunodeficiency viral replication by tannins and related compounds. Among 87 chemically defined tannins and related compounds, several hydrolyzable tannins, but not condensed tannins or other lower molecular weight polyphenols, significantly inhibited both the cytopathic effect of human immunodeficiency virus (HIV) and the expression of HIV antigen in human lymphotropic virus type I (HTLV-1)-positive MT-4 cells. The 50% effective concentrations (2.0-4.8 micrograms/ml) of the active compounds were 13- to 15-fold lower than their 50% cytotoxic concentrations. Their anti-HIV activity was demonstrated to be mediated, at least in part, by inhibition of HIV adsorption to the cells.


Effects of Phyllanthus plant extracts on duck hepatitis B virus in vitro and in vivo. The effects of extracts of five Australian Phyllanthus species (P. hirtellus, P. gunnii, P. gasstroemii, P. similis and P. tenellus), other plant extracts and the antiviral drug foscarnet on duck hepatitis B virus (DHBV) endogenous DNA polymerase (DNAp) activity were compared. All 5 Phyllanthus species caused 50% inhibition at concentrations of dry weight between 350-800 micrograms/ml, which is comparable with the effect described for P. amarus on the DNAp of human and woodchuck hepatitis B viruses. Incubation of P. hirtellus with 100 ID50 DHBV neutralized infection. However, neither P. gasstroemi extract, given by intraperitoneal injection (i.p.) at a dose of 20 mg/kg 3 times per week to ducklings early in the incubation period, or P. hirtellus extract, given to established DHBV carrier ducklings, prevented or eliminated infection.


Topical delivery of liposomally encapsulated gamma-interferon. The extent of uptake of gamma interferon (gamma-IFN) in various strata of hairless mouse, human and hamster skin upon application of a liposomal formulation and an aqueous solution were determined by in vitro diffusion cell experiments. For each of the animal species studied, 70-80% of the liposomally entrapped IFN was deposited onto or penetrated into the skin as determined 24 h after in vitro application. However, a significant fraction of this total amount (approximately 0.25-0.30) is either adsorbed to or associated with the stratum corneum. The drug content found in the deeper skin strata, where the receptor sites reside, suggests that drug deposition is strongly influenced by the skin species tested. The percent of applied drug found in this strata 24 h after application followed the order: hamster (6.1) much greater than human (0.9) greater than hairless mouse (0.3), although the amounts of drug in the total skin of each species tested were approximately the same. This indicates that the deposition of drug into the living epidermis and/or dermis cannot be predicted by determination of the amount of drug in the total skin. The amounts in the deeper skin strata were also in the order of increasing number of follicles/hair in the skin species, suggesting that the transfollicular route is an important pathway for liposomal topical therapeutics.


An ELISA system for evaluating antiretroviral activity against Rauscher murine leukemia virus. A system for evaluating the activity of antiviral agents against Rauscher murine leukemia virus (R-MuLV) has been developed using an enzyme linked immunosorbent assay technique. The activity of various antiviral compounds demonstrated in this assay system has been compared to their activity in the UV-XC plaque reduction assay, which has been used historically for evaluating anti-R-MuLV compounds. The assay is based upon detection of R-MuLV encoded p30 protein production in virus infected murine cells. The assay reagents are readily available and the assay system is amenable to automated data collection systems. Cytotoxicity evaluations are conducted in parallel to the Rauscher MuLV ELISA assay in order to assess drug-induced reductions in cell viability. Cytotoxicity evaluations are important to interpretation of the ELISA results since reductions in cell viability reduce viral protein production which would indicate an antiviral drug effect. This system is less sensitive than the classical UV-XC plaque reduction assay; however, it does offer an alternative to the time-consuming and labor-intensive plaque assay.


A new and potent 2-5A analogue which does not require a 5'-polyphosphate to activate mouse L-cell RNase L. In order to explore the possibility of supplanting the requirement of a 5'-triphosphate moiety for the activation of the 2-5A-dependent endonuclease (RNase L) of mouse L-cells, two new tetrameric analogues of 2-5A were synthesized. The first tetramer, obtained by both a modified prebiotic synthetic approach as well as a phosphite triester solid phase oligonucleotide synthesis method, was p5'A2'p5'A2'p5'(br8A)2'p5'(br8A). The second oligonucleotide was derived from the former by a sequence involving periodate oxidation, reaction with n-hexylamine, and cyanoborohydride reduction, resulting in conversion of the 2'-terminal adenosine residue to 9-(3'-aza-4'-hexyl-1',2',3',4'-tetradeoxyhexopyranos-1(1)-yl)-8-++ +bromoadenine. Both of these oligomers, bearing only 5'-monophosphate groups, were found to be as potent as 2-5A itself as activators of the RNase L of mouse L-cells.


Baculovirus expressed herpes simplex virus type 1 glycoprotein C protects mice from lethal HSV-1 infection. A recombinant baculovirus (vAc-gC1) was constructed that expresses the glycoprotein C (gC) gene of herpes simplex virus type 1 (HSV-1). When Sf9 cells were infected with this recombinant, a protein that was smaller in size than authentic HSV-1 gC was detected by Western blotting using anti-gC polyclonal antibody. The recombinant gC was susceptible to tunicamycin, partially resistant to Endo-H, and was found on the membrane of Sf9 cells. Antibodies raised in mice to recombinant gC reacted with gC from HSV-1 infected cells and neutralized the infectivity of HSV-1 in vitro. Immunized mice were protected from lethal challenge with HSV-1.


Virucidal effect of murine duodenal extracts: studies with lactate dehydrogenase-elevating virus. Mucosal resistance to infection with lactate dehydrogenase-elevating virus (LDV) has been previously demonstrated, and the LDV system presents an important murine model for the study of mucosal barriers to viral infection. In the present study, duodenal molecules were isolated from normal mice which had potent virucidal activity, when tested against LDV as well as canine herpes, canine hepatitis, Semliki forest, and visna viruses. The virucidal activity was demonstrated to be non-immune in nature, and was present in apparently non-enzymatic protein molecules, having a molecular mass of between 10-100 kDa by membrane filtration and 10-17 kDa by gel filtration. The anti-LDV activity of these molecules was suppressed by anti-duodenum antibodies in vitro, and in vivo studies suggested a possible protective role for the anti-viral molecules. We conclude that the normal mouse duodenum contains potent virucidal molecules, which are of interest to the study of biological and molecular mechanisms of viral resistance.


Purification and characterization of the phosphoenolpyruvate carboxylase from the facultative chemolithotroph Thiobacillus novellus (ATCC 8093). Phosphoenolpyruvate carboxylase (EC4.1.1.31), which catalyzes the carboxylation of phosphoenolpyruvate to produce oxaloacetate was purified 465-fold from extracts of organotrophically grown Thiobacillus novellus. Nondenaturing polyacrylamide gel electrophoresis (PAGE) of the purified enzyme revealed the presence of two bands after staining with Buffalo Black. Gels stained with Fast Violet B after incubation with PEP, HCO3-, Mg2+ and acetyl CoA also showed two bands of activity with the faster moving the more active of the two. Sodium dodecylsulfate (SDS)-PAGE of the enzyme heated at 100 degrees C for 5 min revealed the presence of three intensely stained bands of M(r) 95 K, 51 K, and 28 K. However, electrophoresis of the enzyme heated for 2 min showed a single band of about 100 K, indicating that the preparation was likely homogeneous. The 51 K and 28 K subunits are thus products of the 95 K subunit. Gel filtration studies of the native enzyme yielded a M(r) of 360 K. Therefore, the enzyme is a tetramer. The optimum pH in Tris buffer was 8.0, with Km for PEP 0.64 mM, HCO3- 0.11 mM, and acetyl CoA a potent activator, 1.3 microM. A divalent cation best served by Mg2+ gave sigmoidal initial velocity plots. Hill plots of the data gave coefficients (nH) of 2.6. None of the metabolites tested, nucleotide triophosphates excepted, significantly affected enzyme activity. Binding studies with 14C-labelled PEP revealed the binding of about 20 moles PEP per mole (360,000 g) of PEPC. Initial velocity studies suggest that the reaction is catalyzed by a random Bi Bi mechanism. Despite the lack of inhibition by certain metabolites, the enzyme's function is probably anaplerotic.


Isolation of auxotrophic mutants of the methylotrophic yeast Candida boidinii and determination of its ploidy. Auxotrophic mutations in the methylotrophic yeast strain Candida boidinii 11Bh were induced by different mutagens and their combinations (nitrosoguanidine, UV light, HNO2+UV). Majority of the mutants obtained carried defects in histidine, arginine, proline and/or adenine biosynthetic pathways. His- mutants were distributed into four complementation groups using the protoplasts fusion technique. Ploidy determination of Candida boidinii 11Bh was performed by measuring its DNA content and by following its survival after chemical mutagens treatment. The DNA content of this strain was found to be similar to that of a Saccharomyces cerevisiae diploid strain. Also the kinetics of survival of Candida boidinii cells indicate that Candida boidinii 11Bh is a diploid.


Degradation of some phenols and hydroxybenzoates by the imperfect ascomycetous yeasts Candida parapsilosis and Arxula adeninivorans: evidence for an operative gentisate pathway. The imperfect ascomycetous yeasts Candida parapsilosis and Arxula adeninivorans degraded 3-hydroxybenzoic acid via gentisate which was the cleavage substrate. 4-Hydroxybenzoic acid was metabolized via protocatechuate. No cleavage enzyme for the latter was detected. In stead of this NADH- and NADPH-dependent monooxygenases were present. In cells grown at the expense of hydroquinone and 4-hydroxybenzoic acid, enzymes of the hydroxyhydroquinone variant of the 3-oxoadipate pathway were demonstrated, which also took part in the degradation of 2,4-dihydroxybenzoic acid by C. parapsilosis.


Purification and characterization of an NAD(+)-linked formaldehyde dehydrogenase from the facultative RuMP cycle methylotroph Arthrobacter P1. When Arthrobacter P1 is grown on choline, betaine, dimethylglycine or sarcosine, an NAD(+)-dependent formaldehyde dehydrogenase is induced. This formaldehyde dehydrogenase has been purified using ammonium sulphate fractionation, anion exchange- and hydrophobic interaction chromatography. The molecular mass of the native enzyme was 115 kDa +/- 10 kDa. Gel electrophoresis in the presence of sodium dodecyl sulphate indicated that the molecular mass of the subunit was 56 kDa +/- 3 kDa, which is consistent with a dimeric enzyme structure. After ammonium sulphate fractionation the partially purified enzyme required the addition of a reducing reagent in the assay mixture for maximum activity. The enzyme was highly specific for its substrates and the Km values were 0.10 and 0.80 mM for formaldehyde and NAD+, respectively. The enzyme was heat-stable at 50 degrees C for at least 10 min and showed a broad pH optimum of 8.1 to 8.5. The addition of some metal-binding compounds and thiol reagents inhibited the enzyme activity.


Formation of antigenic extracellular polysaccharides by selected strains of Mucor spp., Rhizopus spp., Rhizomucor spp., Absidia corymbifera and Syncephalastrum racemosum. In this study, polyclonal IgG antibodies raised against extracellular polysaccharides (EPS) of Mucor racemosus were characterised as almost specific for moulds belonging to the order of Mucorales. Cross-reactivity in the ELISA could be observed only towards the yeast Pichia membranaefaciens. EPS were isolated from various cultures of M. hiemalis growing on six different carbon sources and two nitrogen sources, with ratios varying from 0.13 to 0.44 relative to the amount of biomass. Other strains including Mucor spp., Rhizopus spp., Rhizomucor spp., Absidia corymbifera and Syncephalastrum racemosum also excreted EPS, with ratios varying from 0.05 to 0.23. In all cases, the excreted EPS had similar antigenic properties as determined by ELISA. No enzymatic degradation of the antigenic parts of the polysaccharides could be observed upon prolonged incubation. Considering that all tested strains formed similar amounts of antigenic EPS there might be scope for the specific detection of biomass of Mucoralean moulds using ELISA techniques for example in food.


Systematic placement of the basidiomycetous yeast Cystofilobasidium lari-marini comb. nov. as predicted by rRNA nucleotide sequence analysis. Based on similarities in basidial morphology and nucleotide sequences of the V3 variable region in the large sub-unit ribosomal RNA, the yeast Leucosporidium lari-marini is considered phylogenetically related to the genus Cystofilobasidium. Therefore the new combination Cystofilobasidium lari-marini is proposed.


Hyalohyphomycosis caused by Paecilomyces variotii: a case report, animal pathogenicity and 'in vitro' sensitivity. A case of cutaneous infection in a 25-year-old male caused by Paecilomyces variotii is described. Animal pathogenicity studies with normal and cortisone-treated mice revealed the predeliction of P. variotii for skin and liver in both normal and cortisone-treated mice and for lungs and heart only in immunosuppressed mice. 5-fluorocytosine gave the best MIC value for P. variotii in vitro. This report documents for the first time that P. variotii causes cutaneous infection.


Heterotrophic nitrification and aerobic denitrification in Alcaligenes faecalis strain TUD. Heterotrophic nitrification and aerobic and anaerobic denitrification by Alcaligenes faecalis strain TUD were studied in continuous cultures under various environmental conditions. Both nitrification and denitrification activities increased with the dilution rate. At dissolved oxygen concentrations above 46% air saturation, hydroxylamine, nitrite and nitrate accumulated, indicating that both the nitrification and denitrification were less efficient. The overall nitrification activity was, however, essentially unaffected by the oxygen concentration. The nitrification rate increased with increasing ammonia concentration, but was lower in the presence of nitrate or nitrite. When present, hydroxylamine, was nitrified preferentially. Relatively low concentrations of acetate caused substrate inhibition (KI = 109 microM acetate). Denitrifying or assimilatory nitrate reductase were not detected, and the copper nitrite reductase, rather than cytochrome cd, was present. Thiosulphate (a potential inhibitor of heterotrophic nitrification) was oxidized by A. faecalis strain TUD, with a maximum oxygen uptake rate of 140-170 nmol O2.min-1.mg prot-1. Comparison of the behaviour of A. faecalis TUD with that of other bacteria capable of heterotrophic nitrification and aerobic denitrification established that the response of these organisms to environmental parameters is not uniform. Similarities were found in their responses to dissolved oxygen concentrations, growth rate and ammonia concentration. However, they differed in their responses to externally supplied nitrite and nitrate.


Kluyveromyces piceae sp. nov., a new yeast species isolated from the rhizosphere of Picea abies (L.) Karst. Two strains of an undescribed species of the genus Kluyveromyces were recovered from the rhizosphere of spruce. A description of the new species, Kluyveromyces piceae, is given and its classification on basis of ascospore shape, substrate utilization, G+C-content, DNA-DNA-reassociation data and habitat specificity is discussed.


Pulmonary autograft. An aortic valve replacement alternative. In selected patients, the pulmonary autograft procedure utilizing cryopreserved homografts for reconstruction of the right ventricular outflow tract is becoming an increasingly popular aortic valve replacement alternative. Longitudinal statistical reports show that patients need reoperation less often with this procedure. Because the valve is autogenous tissue, all indications to date show that the valve continues to function for extended periods of time in all patients and can accommodate growth in children. At the same time, transfer of the pulmonary valve to the aortic position provides a natural valvular flow pattern and freedom from the degenerative changes associated with bioprosthetic valves or the need for anticoagulation associated with mechanical valves.


Development of tolerance to endogenous opiates activated by 24-h food deprivation. Four experiments assessed the effects of exposure to 24-h food deprivation on the tail-flick latency of rats exposed to a temperature stimulus. Confirming previous studies, Experiment 1 showed that food deprivation gave rise to analgesia, as indicated by increased tail-flick latencies, that was antagonized by naloxone. Experiment 2 found that analgesia was greatly reduced after five exposures to periods of 24-h food deprivation (alternating with 24-h free access to food), indicating the development of tolerance. Experiments 3 and 3a examined the development of tolerance to the analgesic effects of morphine following repeated morphine injections, saline injections, and exposure to 24-h food deprivation plus saline injections. The combined results of both experiments provided evidence that repeated exposures to either morphine or food deprivation, produced greater tolerance to morphine than did exposures to saline. That food deprivation was cross-tolerant with morphine indicated that tolerance to food deprivation-induced analgesia involved opioid mechanisms. The relevance of opioid tolerance to psychobiological models of feeding and to the development of an animal model of anorexia nervosa was discussed.


Factors influencing sensory-specific satiety. In the first part of this study, subjects tasted small portions of a set of foods (rating set) and rated their liking of these foods before and after eating a serving of one of the foods (test meal). These foods were chosen to vary in both macronutrient composition and sensory qualities. Measurements of hunger were taken before and after the test meal. The amount of sensory-specific satiety produced by a test meal differed depending on the food eaten. There was a trend for high-protein foods, which were also the least-liked foods, to decrease more in liking than low-protein foods. Buttered rolls and Coke, when eaten as test meals, dropped the least in liking. Initial liking and the variety of sensory qualities within a food were investigated as potential factors which could influence sensory-specific satiety. Subjects tasted and rated their liking of the test meals, which varied in the level of initial liking and the level of variety of sensory qualities within a food, before and after eating. There was a non-significant trend for the less-liked test meals to drop more in liking than the well-liked test meals. There was also a slight trend for the low-variety test meals to drop slightly more in liking than the intermediate and high-variety test meals.


Predictors of adult humans' self-control and impulsiveness for food reinforcers. Humans vary in the degree to which they demonstrate self-control--choice of larger, more delayed over smaller, less delayed reinforcers. When reinforcers consist of food, individual human subjects' choice behavior varies from virtually exclusive self-control to exclusive impulsiveness. The present experiment, using 26 men and 26 women subjects, explored some possible sources of this individual variation through assessing the correlation of behaviors exhibited in the self-control paradigm with various subject characteristics. The results suggest that self-control is negatively related to individuals' reported susceptibility to hunger (Factor 3 of the Three-Factor Eating Questionnaire) and positively related to overall rating of juice desirability, but not to gender or to various measures of caloric deprivation or personality. Relatively high self-perceived levels of food deprivation may decrease self-control, whereas assigning a relatively high value to the reinforcer may increase self-control. Both tendencies would have been adaptive for evolving humans.


Methodology and theory in human eating research. Current research in human eating is assessed from the perspective of current research methods which stress laboratory research, the use of artificial foods rather than real meals, shorter term studies, animal models, and abnormal eating models and an emphasis on sensory and physiological factors rather than social, cultural and contextual factors. The proposal is made to refocus more human eating research on real people eating real foods in real eating situations.


Where should human eating be studied and what should be measured? Meiselman's (1992) proposal for redirection of research on human eating behavior towards eating in real settings is misdirected, because it does not take into consideration the questions being addressed by laboratory studies. In fact many of the studies for which he has called have in fact been done and will be cited. There is no compelling scientific or strategic reason that investigators who are making good progress on the problem of what controls the amount eaten should be exhorted to examine what controls the place or time of eating or what is selected for eating. They are different problems which require different methodology. The field is large enough for both kinds of studies and there is no reason to berate investigators as Meiselman does for not investigating the problem he happens to be studying.


Kinetics and activity distribution of urease coencapsulated with hemoglobin within polyamide membranes. A 91.5% mass yield of urease and hemoglobin (Hb), co-encapsulated within polyamide membranes, was determined spectrophotometrically. The specific activity yield of microencapsulation was 84%, twofold higher than values previously reported, as a result of optimization of encapsulation conditions. The kinetic parameters and pH activity profiles of intracapsular urease were determined to be similar to those corresponding to the free enzyme. Similar activities were also observed for intact and microcapsule homogenate, indicating minimal mass transfer and diffusional limitation. The active configuration of the enzyme appears to remain intact upon microencapsulation. The application of a kinetic model for encapsulated urease further indicated that the kinetics were reaction-controlled with minimal mass transfer restrictions.


Activation and detection of (pro)mutagenic chemicals using recombinant strains of Streptomyces griseus. Two recombinant strains of Streptomyces griseus have been developed to report on the activation of promutagenic chemicals. This activation is monitored by reversion of the bacterial test strains to a kanamycin-resistant phenotype. Strain H69 detects point mutations and was reverted at an increased frequency by acetonitrile, 2-aminoanthracene, 1,2-benzanthracene, benzidine, benzo(a)pyrene, 9,10-dimethyl-1,2-benzanthracene, and glycine. The second strain, FS2, detects frame shift mutations and was reverted at an increased frequency by 1,2-benzanthracene, benzidine, and glycine. Compounds such as butylated hydroxytoluene, catechol, chlorobenzene, hydroquinone, potassium chloride, phenol, cis-stilbene, trans-stilbene, and toluene did not elicit positive responses in either strain. In addition, these strains are capable of detecting direct-acting mutagens such as N-methyl-N'-nitrosoguanidine and ICR-191, providing further evidence of their promise for detecting a wider range of mutagens. To our knowledge, this is the first report of bacterial strains capable of activating promutagenic compounds and detecting their mutagenic metabolites without the benefit of an exogenous activation system such as the rodent liver homogenate (S9).


Actinomycetes in submerged culture.

Actinomycetes in submerged culture. The range of morphological forms of actinomycetes in shaken flask culture and fermenters is reviewed. Some of the factors that influence pellet formation and its prevention are discussed. The relationship between morphology and production of antibiotics and other metabolites is also examined.


Rapid determination of immobilized ConA lectin activity. ConA was immobilized on an epoxy-activated copolymer of 2-hydroxyethyl-methacrylate and ethylene-dimethacrylate and commercially available high-pressure liquid chromatography (HPLC) sorbents Separon HEMA 1000 EL, Separon HEMA 1000 E, and Separon HEMA 1000 EH (Tessek, Prague, CSFR Denmark). Specific, sensitive, and rapid method for determination of immobilized ConA lectin activity was developed. beta-Galactosidase from Aspergilus oryzae oligomannosyl residues was used as specific affinant. After separation of bound and unbound beta-galactosidase, enzyme activity was measured in supernatant and thus immobilized ConA lectin activity was calculated easily. The use of the method for evaluating the properties of immobilized ConA, efficiency of immobilization, specific activity, and thermostability is shown. The method developed could be generalized by using artificially glycosylated enzyme for any lectin.


Glucoamylase immobilization on a magnetic microparticle for the continuous hydrolysis of maltodextrin in a fluidized bed reactor. Glucoamylase (GA) has been successfully immobilized through its carbohydrates previously oxidized with periodate onto a low-cost magnetic microparticle made of polyethyleneimine-coated magnetite crosslinked with glutaraldehyde (M-GAD) and derivatized with adipic dihydrazide (ADH). A stabilization posttreatment consisting of crosslinking its carbohydrates with ADH, increased the remaining activity from 54 to 71%, calculated on the Vm values and measured at 50 degrees C and pH 4.5 with maltodextrin (DE 11-14) as substrate. This treatment also improved the enzyme stability and lowered the deactivation rate constant kd to a third of its value. A 30% maltodextrin solution has been continuously hydrolyzed at 50 degrees C and pH 4.5 in a recycled, fluidized bed reactor (FBR) containing GA immobilized on these magnetic microparticles. They easily settled in this highly viscous medium because of their high density (5 g/mL), and washout of ultrafines was prevented by surrounding the top of the bed with an electromagnet. The small particle size (20 microns) allowed a high enzyme loading in the reactor and also a high bed voidage, which is recommended to avoid extensive pressure drop and consequent channeling problems. The kinetic of hydrolysis fitted with the plug-flow model; this is explained by the insignificant backmixing effects observed. After 2 wk of hydrolysis under process conditions leading to a conversion of 70%, which corresponds to a high-conversion syrup, the immobilized GA only lost 4% of its initial activity.


Ascorbic acid biosensor using ascorbate oxidase immobilized on alkylamine glass beads. A biosensor for ascorbic acid based on enzyme kinetics of ascorbate oxidase (E.C.1.10.3.3) was developed. The enzyme was extracted from Cucurbita maxima, or jerimun and immobilized by covalent bounding, using glutaradehyde as a bifunctional agent, on alkylamine glass beads, with and without enzyme active site protection. A low-cost, home-made oxygen electrode was applied as a transducer. The system has sensitivity from 62.5 up to 500 microM of ascorbic acid with satisfactory operation for more than 2 mo.


Biological deodorization of dimethyl sulfide using different fabrics as the carriers of microorganisms. Biological deodorization of dimethyl sulfide (DMS) was studied using nine unwoven fabrics as the carriers of microorganisms in a laboratory-scale deodorizing system. The activated carbon fabric FN-200CF-15 was the best packing material compared with other packing materials used, on the basis of removal rate. The maximum removal rate (Vm), evaluated by using Michaelis-Menten equation, was 2.28 g-S.kg-dry fab.-1.day-1 in this fabric biofilter. The critical load of DMS in this fabric biofilter was dependent on space velocity (SV), determined as 0.78 and 0.66 g-S.kg-dry fab.-1 at SV 100 and 150 h-1, respectively. Strain IM1 isolated from the carbon fabric FN-200CF-15 biofilter in modified Waksman (MW) medium successfully degraded DMS as well as hydrogen sulfide (H2S), methanethiol (MT), and dimethyl disulfide (DMDS) in batch test. The DMS removal rates (g-S.cell-1.h-1) by this strain measured in batch culture and calculated in FN-200CF-15 biofilter by the cell numbers appeared in MW medium were found almost equal, indicating that strain IM1 may be the dominant microorganism in this biofilter.


Enzymatic synthesis of (R) and (S) 1-deuterohexanol. This paper describes practical enzymatic procedures for the synthesis of (R) and (S) 1-deuterohexanol, a useful building block for chiral poly isocyanated liquid crystals. Alcohol dehydrogenases from horse liver and Pseudomonas catalyzed the reduction of hexanal with deuterated NAD (NADD) resulting in 50% and 89% yields of (R) and (S) 1-deuterohexanol, respectively. The deuterated cofactor was regenerated in situ by alcohol dehydrogenase catalyzed oxidation of ethanol-d6 or 2-propanol-d8. The (S) alcohol was also synthesized by the horse liver alcohol dehydrogenase reduction of 1-deuterohexanal, which was prepared chemically from hexanal. The yields of the reaction were greatly increased by the use of a biphasic system or with the immobilized enzyme in anhydrous organic solvents. Horse liver alcohol dehydrogenase was stabilized by immobilization on PAN or noncovalent entrapment on XAD resin.


The study of the cultivation of Chinese hamster ovary and Bowes cell lines with poly(organophosphazene) membranes. Sixteen poly(organophosphazenes) were prepared by reaction between polydichlorophosphazene (NPCl2)n and nucleophilic reagents such as phenoxides and amino compounds. Adhesive and colony formation percent were investigated using V-79 Chinese hamster cells on poly(organophosphazene) films. It was found that [NP(OC6H5)2-x(NHBu-n)x]n (x = 0.2, 1.8) gave the best percent adhesiveness. This value was similar to that of Falcon. On the other hand, [NP(OC6H5)-(NHBu-n)]n film showed best colony percent formation. This was of a higher value than that of Falcon. The [NP(OC6H5)2]n properties were poor for cultivation of useful Bowes and chinese hamster ovary cell lines in comparison with Cytodex III.


Immobilization of glucose isomerase onto granular chicken bone. Glucose isomerase was immobilized onto granular chicken bone (BIOBONE) by adsorption. The amount of activity bound relative to an equal amount of free enzyme was 32 +/- 1%, with the estimated specific activity decreasing from 11.1 +/- 0.7 to 3.9 +/- 0.5 U/mg protein with immobilization. Compared with the free enzyme, immobilized glucose isomerase showed a threefold increase in the Km for fructose and a fivefold decrease in Vmax. High operating temperatures were possible (greater than 55 degrees C), but continuous use and long-term storage studies showed gradual losses of activity. Both the binding and the activity of the bone-immobilized enzyme were highly resistant to treatments with detergent, ethanol, and KCl. Studies to determine mass transfer limitation effects on immobilized glucose isomerase showed that these were insignificant for this system.


Psychosocial responses among individuals with human immunodeficiency virus infection. The purpose of this research was to identify and describe psychosocial responses to being infected with human immunodeficiency virus (HIV). Ten adults infected with HIV were interviewed and audiotaped as they described their feelings and experiences as HIV-infected people. The interviews were analyzed using a grounded theory methodology. The core category that emerged from the data was Fighting to Survive with HIV Infection. Supporting concepts were Taking Care and Restructuring One's Life. The concept of Taking Care included processes of everyday work and illness work. The processes involved in Restructuring One's Life were living with uncertainty, accepting homosexuality, experiencing changing feelings, protecting confidentiality, dealing with the medical profession, handling multiple losses, and living with a terminal illness.


Immobilization of amyloglucosidase onto granular chicken bone. Amyloglucosidase was immobilized onto granular chicken bone (BIOBONE) by noncovalent interactions. The amount of activity bound relative to an equal amount of free enzyme was 13.6 +/- 0.4%. The estimated specific activity for amyloglucosidase decreased from 75.3 +/- 0.8 to 43.5 +/- 9.6 U/mg protein upon immobilization. The Km value of the bone-immobilized enzyme using glycogen as substrate increased from 3.04 +/- 0.38 mg/mL (free) to 9.04 +/- 1.51 mg/mL (immobilized), but Km showed no change upon immobilization when starches were used as substrates. A decrease in Vmax values occurred upon enzyme immobilization for all substrates, but this largely reflected the percentage of enzyme initially bound to the bone. Immobilization also improved enzyme stability in the presence of various additives (e.g., detergent, KCl, and ethanol) or under low or high pH reaction conditions. Bound amyloglucosidase maintained high activity (greater than 90%) following five cycles of continuous use at moderate (23 degrees C) and high (55 degrees C) temperatures. Data derived from Lineweaver-Burk and Arrhenius plots indicated that substrate and product diffusion limitation were minimal.


Home health nurses' assessments of cognition. Assessments of mental status in elderly persons admitted to home health agencies are the basis for decision-making related to resources provided. A home health nurse's perceptions of an elderly client's capacity for self-care determines whether community services will be prescribed. In institutional settings, evaluation of cognition by professional nurses is often incomplete or inaccurate. It was the purpose of this study to compare the judgements of cognition made by nine home health nurses who were admitting elderly clients to a visiting nurse association with scores from an objective measure of mental status. Findings suggest that these nurses relied primarily on orientation as an indicator of cognitive abilities.


Preoperative self-efficacy and postoperative behaviors. Preoperative instruction has been found to decrease postoperative complications; however, the mechanisms through which preoperative instruction exerts its effects remain unknown. In this study, the relationship between preoperative self-efficacy and postoperative behaviors was investigated. Subjects were 68 cholecystectomy patients. Subjects' scores on the efficacy expectations subscale were related significantly to postoperative deep breathing, ambulation, and recall of expected events; scores on the outcome expectations subscale were related significantly to postoperative requests for pain medication. Self-efficacy theory provided a link between self perceptions and behavior and may be the mechanism through which preoperative instruction exerts its effects.


Determining risk status in a primary care setting. Nurses working in primary health care settings often care for large numbers of clients in brief periods of time. Both clients and nurses express frustration toward the care provided in these circumstances. Development of a screening tool to identify high-risk clients could assist nurses in targeting interventions to these individuals; in turn, this has the potential for increasing nurse and client satisfaction. A general procedure for identifying persons at risk in primary health care settings is described. The procedure is illustrated with a specific population of clients--incarcerated women. Factors found important in assessing risk were social characteristics such as education, situational factors such as sentence length, and indicators of psychological distress such as depression. These broad categories may serve as a basis for the development of screening tools for a variety of populations.


Expression of mouse cathepsin L cDNA in Saccharomyces cerevisiae: evidence that cathepsin L is sorted for targeting to yeast vacuole. To investigate the intracellular transport mechanism of lysosomal cathepsin L in yeast cells, we attempted to produce mouse cathepsin L in Saccharomyces cerevisiae by placing the coding region under the control of the promoter of the yeast glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene. Immunoblotting analysis by the use of an antibody specific for rat cathepsin L revealed that the yeast cells carrying the cathepsin L coding sequence produced 39- and 30-kDa products, which correspond to the rat procathepsin L and the single-chain form of mature cathepsin L, respectively. The precursor polypeptide showed sensitivity toward endoglycosidase H treatment. Cell fractionation experiments demonstrated that the processed form of 30-kDa cathepsin L was found to be colocalized to the yeast vacuole with the marker enzyme carboxypeptidase Y in a Ficoll step gradient. In the prepared vacuolar fraction, a considerable amount of cathepsin L was revealed to be cofractionated with the vacuolar membranes. Furthermore, the phase separation experiments with Triton X-114 provide the first evidence showing that the mature form of cathepsin L polypeptide is strongly associated with the vacuolar membranes. Therefore, the present results suggest that the mouse cathepsin L precursor polypeptide is initially synthesized as the proenzyme in the yeast cells and then correctly delivered to the vacuole. During the intracellular sorting pathway, the procathepsin L would undergo the post-translational proteolytic processing step to generate the mature enzyme. Based on these lines of evidence, we propose that cathepsin L is recognized by mechanisms similar to those for the intracellular sorting and processing of vacuolar proteins in the yeast cells.


Slow-onset inhibition of ribosomal peptidyltransferase by lincomycin. In a system derived from Escherichia coli, we carried out a detailed kinetic analysis of the inhibition of the puromycin reaction by lincomycin. N-Acetylphenylalanyl-tRNA (Ac-Phe-tRNA; the donor) reacts with excess puromycin (S) according to reaction [1], C+S Ks <--> CS k3 --> C'+P, where C is the Ac-Phe-tRNA-poly(U)-ribosome ternary complex (complex C). The entire course of reaction [1] appears as a straight line when the reaction is analyzed as pseudo-first-order and the data are plotted in a logarithmic form (logarithmic time plot). The slope of this straight line gives the apparent ksobs = k3[S]/(Ks + [S]). In the presence of lincomycin the logarithmic time plot is not a straight line, but becomes biphasic, giving an early slope (ke = k3[S]/(Ks(1 + [I]/Ki) + [S])) and a late slope (k1 = k3[S]/(Ks(1 + [I]/K'i + [S])). Kinetic analysis of the early slopes at various concentrations of S and I shows competitive inhibition with Ki = 10.0 microM. The late slopes also give competitive inhibition with a distinct inhibition constant K'i = 2.0 microM. Excluding alternative models, the two phases of inhibition are compatible with a model in which reaction [1] is coupled with reaction [2], C+I k4 <--> k5 CI k6 <--> k7 C*I, where the isomerization step CI <--> CI* is slower than the first step C+I <--> CI, Ki = k5/k4 and K'i = Ki [k7/(k6 + k7)]. Corroborative evidence for this model comes from the examination of reaction [2] alone in the absence of S. This reaction is analyzed as pseudo-first-order going toward equilibrium with kIeq = k7 + (k6 [I]/(Ki + [I])). The plot of kIeq versus [I] is not linear. This plot supports the two-step mechanism of reaction [2] in which k6 = 5.2 min-1 and k7 = 1.3 min-1. This is the first example of slow-onset inhibition of ribosomal peptidyltransferase which follows a simple model leading to the determination of the isomerization constants k6 and k7. We suggest that lincomycin inhibits protein synthesis by binding initially to the ribosome in competition with aminoacyl-tRNA. Subsequently, as a result of a conformational change, an isomerization occurs (CI <--> C*I), after which lincomycin continues to interfere with the binding of aminoacyl-tRNA to the isomerized complex.


Phosphorylation of chicken oviduct progesterone receptor by cAMP-dependent protein kinase. Phosphorylation of immunopurified chicken oviduct progesterone receptor (PR) was studied in intact cells and under cell-free conditions. Cytosol PR was isolated by incubation with anti-PR monoclonal antibody alpha PR22 adsorbed to protein A-Sepharose and suspended in a reaction mixture containing 10 mM Mg2+, 0.1 mM [gamma-32P]ATP, and the catalytic subunit of cAMP-dependent protein kinase (cAMP-PK) from bovine heart. All three major proteins of avian PR (PR-A, 79 kDa; PR-B, 110 kDa; 90 kDa) incorporated 32P-radioactivity on serine residues. The phosphorylation reaction was inhibited by synthetic inhibitors of protein kinases, H-8 and 20-residue peptide IP20. A 40 degrees C preexposure of PR oligomer increased phosphorylation of the 90-kDa protein, known to be a heat-shock protein (hsp-90). The extent of the phosphorylation reaction was temperature-dependent as the 32P-incorporation into PR-A and PR-B increased gradually, showing a maximum at 37 degrees C. Multiple phosphopeptides (4-7) were resolved by two-dimensional electrophoresis chromatography following cleavage of 32P-labeled peptides with trypsin. Both A and B forms of receptor showed similar phosphorylation patterns with B receptor digestion exhibiting two to three additional peptides. Under physiological conditions, preincubation of oviduct mince with forskolin, a regulator of intracellular cAMP levels, caused a greater extent of phosphorylation of PR-A and PR-B proteins. The results of this study demonstrate that chicken oviduct PR is an excellent substrate for the action of cAMP-PK in vitro and that this enzyme may be a physiological regulator of progesterone action in the oviduct.


Analysis of catalytic properties of hen egg white lysozyme during renaturation from denatured and reduced material. Catalytic properties of hen egg white lysozyme were analyzed during the renaturation of the enzyme from completely reduced and denatured material. The formation of intermediate folding products and the generation of native lysozyme was monitored by acetic acid/urea electrophoresis. The results showed that during the beginning of renaturation almost all reduced and denatured lysozyme is converted to forms possessing lower compactness than native lysozyme, probably as a result of formation of only one or two disulfide bonds. Kinetic analysis of lysozyme during renaturation showed that the generation of lysozyme with four disulfide bonds was not necessarily equivalent to the formation lysozyme with native-like catalytic properties. It appeared that the formation rate of the structures of the structures of the substrate binding site and of the catalytic site were limited by the generation of four disulfide bonds containing lysozyme. The catalytic properties of intermediate folding products made it evident that the final structures of the substrate binding site and of the catalytic site were formed after the generation of all disulfide bonds.


Cleavage of xyloglucan by nasturtium seed xyloglucanase and transglycosylation to xyloglucan subunit oligosaccharides. Oligosaccharide subunits were prepared from xyloglucan (XG) by partial hydrolysis with cellulase and added back at micro- to millimolar concentrations to XG in the presence of nasturtium seed xyloglucanase (XG-ase). The oligosaccharides (0.2 mM) stimulated the capacity of this XG-ase to reduce the viscosity of XG solutions by 10- to 20-fold. Purification and fractionation of seed XG-ase activity by gel permeation fast protein liquid chromatography produced a single peak that was much more active in the presence than absence of added XG oligosaccharide. [14C]Fucose-labeled XG nonasaccharide was synthesized by pea fucosyltransferase and shown to be incorporated into polymeric XG in the presence of seed XG-ase without the net production of new reducing chain ends, even while the loss of XG viscosity and XG depolymerization were enhanced. It is concluded that in vitro seed XG-ase can transfer cleavage products of XG to XG oligosaccharides via endotransglycosylation reactions, thereby reducing XG M(r) without hydrolysis. Since this is the only XG-cleaving enzyme that develops in nasturtium seeds during germination, it may be that its transglycosylase and hydrolase capacities are both necessary to account for the rapid and complete depolymerization of XG that takes place.


Effects of MgCl2 on the release and recycling of heparan sulfate proteoglycans in a rat parathyroid cell line. Divalent cations, such as Mg2+, Ba2+, and Co2+, are known to mimic the effects of Ca2+ in parathyroid cells, but it is not clear whether the mechanism of their action is the same as that of Ca2+. We have shown that extracellular Ca2+ concentration ([Ca2+]e) regulates the distribution and recycling of cell-surface heparan sulfate (HS) proteoglycans in a rat parathyroid cell line; at normal to high [Ca2+]e (e.g., 2 mM) HS proteoglycans are primarily localized intracellularly, while at low [Ca2+]e (0.05 mM) they are translocated to the cell surface and rapidly recycle (Takeuchi, Y., Sakaguchi, K., Yanagishita, M., Aurbach, G. D., and Hascall, V. C., 1990, J. Biol. Chem. 265, 13661-13668). We now show that a high concentration of Mg2+ (8 mM) reduces the amount of recycling HS proteoglycans in low [Ca2+]e. However, the primary effects of high Ca2+ and high Mg2+ on the recycling HS proteoglycans are different. High [Ca2+]e causes translocation of HS proteoglycans to intracellular compartments, while high Mg2+ stimulates cleavage of their core proteins and subsequent shedding of HS proteoglycans into the medium, thereby depleting the recycling molecules. However, high Mg2+ does not induce shedding of HS proteoglycans in high [Ca2+]e. The effects of Ba2+ and Co2+ were similar to those of Mg2+, but Sr2+ showed no significant effects on HS proteoglycan translocation. Otherwise, 8 mM Mg2+ did not alter biosynthesis or intracellular catabolism of HS proteoglycans. These observations suggest that the recycling of HS proteoglycans in parathyroid cells is sensitive only to [Ca2+]e, whereas several other divalent cations can deplete the recycling HS proteoglycans by a distinctly different mechanism. Thus, the mechanism by which Ca2+ regulates the amounts of the recycling HS proteoglycans may be more physiological and play a functional role in parathyroid cells.


Comparative study of monomeric reconstituted and membrane microsomal monooxygenase systems of the rabbit liver. I. Properties of NADPH-cytochrome P450 reductase and cytochrome P450 LM2 (2B4) monomers. Oligomers and monomers of NADPH-cytochrome P450 reductase and cytochrome P450 LM2 (2B4) isolated from the liver microsomes of phenobarbital-treated rabbits were examined for physicochemical properties and catalytic activities. As measured using laser correlation spectroscopy the particle sizes of NADPH-cytochrome P450 reductase and cytochrome P450 LM2 oligomers were 14.8 +/- 1.7 and 19.2 +/- 1.4 nm, respectively. Twenty-four-hour incubation with Emulgen 913 at 4 degrees C at a molar ratio of 1:100 led to the monomerization of NADPH-cytochrome P450 reductase and cytochrome P450 LM2 oligomers, the particle sizes diminishing to 6.1 +/- 1.3 and 5.2 +/- 0.4 nm, respectively. The thermal stability of NADPH-cytochrome P450 reductase monomers was the same as that of oligomers, whereas cytochrome P450 LM2 monomers were less thermostable than oligomers and cytochrome P450 in microsomes. Similar to cytochrome P450 LM2 oligomers and the microsomal hemoprotein, cytochrome P450 LM2 monomers formed complexes with type I and II substrates, but with Kd values higher than those of microsomes and cytochrome P450 LM2 oligomers. Kinetic parameters (Vmax and Km) of H2O2- and cumene hydroperoxide-dependent oxidation of benzphetamine and aniline in the presence of cytochrome P450 LM2 oligomers, monomers, and microsomes were determined. Peroxidase activities of the oligomers and monomers were the same, but were lower than those of microsomes. Thus the substitution of protein-protein interactions in cytochrome P450 LM2 oligomers with protein-detergent interactions in the monomers did not influence the catalytic properties of the hemoprotein.


Comparative study of monomeric reconstituted and membrane microsomal monooxygenase systems of the rabbit liver. II. Kinetic parameters of reductase and monooxygenase reactions. The kinetic parameters of NADPH-dependent cytochrome P450 LM2 (2B4) reduction and substrate oxidation in the monomeric reconstituted system, consisting of purified NADPH-cytochrome P450 reductase and cytochrome P450 LM2 monomers, and in phenobarbital-induced rabbit liver microsomes were compared. In the absence of benzphetamine, NADPH-dependent reduction of cytochrome P450 LM2 was monophasic in the monomeric reconstituted system and biphasic in the microsomes. The presence of the substrate in the monomeric reconstituted system caused the appearance of the fast phase. In this system substrate-free cytochrome P450 LM2 was entirely low-spin, and the addition of benzphetamine shifted the spin equilibrium to a high state very weakly. No correlation between high-spin content and the proportion of the fast phase of NADPH-dependent LM2 reduction was found in the system. Vmax values for the oxidation of type I substrates (benzphetamine, dimethylaniline, aminopyrine) in the monomeric reconstituted system were higher or the same as in the microsomes, whereas Km values for the substrates and NADPH were lower in the microsomes. Maximal activity of the monomeric reconstituted system was observed at a 1:1 NADPH-cytochrome P450 reductase/cytochrome P450 LM2 ratio. Measurements of benzphetamine oxidation as a function of NADPH-cytochrome P450 reductase/cytochrome P450 LM2 ratio at a constant total protein concentration allowed the Kd of the NADPH-cytochrome P450 reductase/cytochrome P450 LM2 complex to be estimated as 6.4 +/- 0.5 microM. Complex formation between the NADPH-cytochrome P450 reductase and cytochrome P450 LM2 monomers was not detected by recording the difference binding spectra of the reductase monomers with LM2 monomers or by treatment the mixture of the monomers of the proteins with the crosslinking reagent, water-soluble carbodiimide.


Thermal denaturation of plastocyanin: the effect of oxidation state, reductants, and anaerobicity. The thermal stability of plastocyanin (PC) was determined as a function of oxidation state of the copper center and the presence of oxidants, reductants, oxygen, and EDTA. It was found that the copper center and its ligands play a crucial role in maintaining the stability of PC. Thermal denaturation was monitored by using far-uv circular dichroism (CD) spectra to monitor changes in secondary structure, the near-uv CD ellipticity at 280 nm to monitor changes in tertiary structure, and the absorbance at 597 nm and the 255-nm CD transition to monitor changes in the copper center. Reduced PC (Tm = 71 degrees C) was found to be more stable than the oxidized form (Tm = 61 degrees C). The Tm was increased by addition of reductants, removal of oxygen, or addition of EDTA. Two distinct denatured forms (designated D1 and D2) were separated by anion exchange fast protein liquid chromatography. Neither form contained a native copper center. Form D2 retained the characteristic 280-nm CD band but showed an altered far-uv CD spectrum. Its formation was inhibited by the addition of reductants or the removal of oxygen. It could be refolded to form native, Cu-PC upon incubation with copper plus a reductant such as dithionite. These results suggest that its formation involves the reversible oxidation of a group on the PC molecule, possibly a ligand to the copper such as Cys 84 or Met 92. Form D1 occurred in the presence of ferricyanide or at high temperatures in the presence of oxygen. EDTA inhibited its formation. Form D1 lost the 280-nm CD transition and its far-uv CD spectrum was altered. No renaturation was observed suggesting that Form D1 is the product of an irreversible oxidation step possibly involving a histidine ligand to the copper. Forms D1 and D2 are not interconvertible and represent the endpoints of two different denaturation pathways.