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[Sulfamethoxazole-trimethoprim-induced pneumonitis in a patient with hemophilia B who was infected with the human immunodeficiency virus].

Severe cellular immunosuppression developed in a 25-year-old man with hemophilia B who was infected with the human immunodeficiency virus (HIV). Four days after administration of sulfamethoxazole-trimethoprim (SMX-TMP) for prophylaxis against Pneumocystis carinii pneumonia (PCP), diffuse uptake of both lungs was confirmed on a 67Ga scintigram. Reticular shadows were also seen throughout both lung fields on a chest CT scan. These findings were compatible with PCP, according to the guidelines for presumptive diagnosis of the acquired immunodeficiency syndrome, published by the Centers for Disease Control and Prevention. The dose of SMX-TMP was increased, but interstitial pneumonitis worsened and was accompanied by fever, skin rash, and liver dysfunction, which are common in HIV-infected patients receiving SMX-TMP. No evidence of PCP or of any other opportunistic infection was found by bronchoalveolar lavage. Adverse reactions diminished after SMX-TMP administration was stopped. The 67Ga scintigram and chest CT findings also returned to normal. We concluded that the interstitial pneumonitis was induced by SMX-TMP. SMX-TMP is the first choice anti-PCP drug, but a high incidence of adverse reactions in patients with HIV infection has been reported. Therefore the possibility of SMX-TMP-related pulmonary toxicity must be considered in HIV-infected patients.

AIDS-Related Opportunistic Infections↗

[Effect of tetramethyl pyrazine on coronary vasoconstriction induced by endothelin-1 in dogs].

The purpose of this study was to determine the antagonistic effect of tetramethyl pyrazine (TMP), a sort of chinese herbal medicine, on coronary vasoconstriction induced by endothelin-1 (ET-1) in closed chest dogs. ET-1 at doses of 50, 75 and 100 pmol was selectively administered into left main coronary artery and coronary angiogram was performed in 1, 3 and 10 minutes after intracoronary administration of ET-1. After a 60 minute interval ET-1 administration and coronary angiogram were repeated in two groups in group A with 5 dogs intravenous infusion of saline solution was administered while in group B with 4 dogs TMP was infused at a dose of 80 mg/kg. Blood pressure of intra-femoral artery, heart rate and ECG were monitored during the experiment. The study demonstrated that coronary vessel diameter significantly decreased by 17% (P < 0.02) in group A and 20% (P < 0.02) in group B, associated with ischemia in ECG (4/5 in group A and 3/4 in group B) after intracoronary administration of ET-1. Endothelin-1 induced coronary vasoconstriction and ischemic changes in ECG were significantly inhibited by intravenous TMP. The coronary diameter increased by 20% (P < 0.03) after administration of TMP, comparing with the control group. Heart rate had an increased response to TMP. In conclusion this study demonstrated that intracoronary administration of ET-1 caused significant myocardial ischemia through coronary vasoconstriction, which was inhibited by TMP. TMP significantly dilated coronary artery.

Animals↗

A single amino acid substitution in Staphylococcus aureus dihydrofolate reductase determines trimethoprim resistance.

A single amino acid substitution, Phe98 to Tyr98, in dihydrofolate reductase (DHFR) is the molecular origin of trimethoprim (TMP) resistance in Staphylococcus aureus. This active site amino acid substitution was found in all S. aureus TMP-resistant clinical isolates tested. In order to explore the structural role of Tyr98 in TMP-resistance the ternary complexes of the chromosomal S. aureus DHFR (SaDHFR) with methotrexate (MTX) and TMP in the presence of nicotinamide adenine dinucleotide phosphate (NADPH) as well as that of mutant Phe98Tyr DHFR SaDHFR(F98Y) ternary folate-NADPH complex have been determined by X-ray crystallography. Critical evidence concerning the resistance mechanism has also been provided by NMR spectral analyses of 15N-labelled TMP in the ternary complexes of both wild-type and mutant enzyme. These studies show that the mutation results in loss of a hydrogen bond between the 4-amino group of TMP and the carbonyl oxygen of Leu5. This mechanism of resistance is predominant in both transferable plasmid-encoded and non-transferable chromosomally encoded resistance. Knowledge of the resistance mechanism at a molecular level could help in the design of antibacterials active against multi-resistant Staphylococcus aureus (MRSA), one of todays most serious problems in clinical infectology.

Binding Sites↗

Neonatal death and lung injury in rats caused by intrauterine exposure to O,O,S-trimethylphosphorothioate.

O,O,S-Trimethyl phosphorothioate (OOS-TMP) is an impurity present in a number of widely used organophosphorus insecticides and has been recognized as a potent lung toxicant. OOS-TMP was given p.o. to pregnant rats on gestation day (G) 20 at 0.5, 2.5, 10 and 40 mg/kg. Control dams or pair-fed dams (pair-fed to 40 mg/kg) received 2 ml/kg corn oil. Neonates from treated dams died within 72 h after delivery in a dose-related manner: 100% at 40 mg/kg, 86% at 10 mg/kg, 15% at 2.5 mg/kg, 1% at 0.5 mg/kg, with 3% in controls and 2% in neonates from pair-fed dams. Neonates from treated (40 or 10 mg/kg) and control dams were cross-fostered. The cross-fostering did not affect mortality of neonates from either dosed dams or from control dams. Disposition of OOS-TMP was studied by using [3H]-OOS-TMP at 0.5, 2.5 and 10 mg/kg. Concentrations of OOS-TMP equivalent in fetal lung were about one half of those in mothers at all doses. In another set of experiments, dams (five dams for each dose) were dosed on G 20 with OOS-TMP p.o. at 0, 0.5, 2.5, 10, and 40 mg/kg or pair-fed (pair-fed to 40 mg/kg) and the fetuses were delivered by cesarean section (C-section) on G 23. In neonates from dams dosed with 10 and 40 mg/kg, cyanosis occurred within 4 h after C-section. Histopathological examination revealed dose-related proliferation of type II pneumocytes in dams and proliferation of interstitial cells and delayed septal/capillary development in neonates.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

Pharmacokinetic study of the new sulfamethopyrazine-trimethoprim combination (kelfiprim) in renal insufficiency.

The combination of trimethoprim (TMP) and sulfamethopyrazine (SMP) has been successfully used to treat chronic urinary tract infections. Since parenchymal involvement associated with renal insufficiency of varying degree is not infrequent in these patients, it was considered important to study the pharmacokinetics of TMP and SMP in a fixed dose combination. Four groups of patients were studied: 1) 4 patients with endogenous creatinine clearance (CLcR) between 80 and 40 ml/min; 2) 3 patients with CLcR between 40 and 10 ml/min; 3) 3 patients on chronic peritoneal dialysis (CAPD); and 4) 3 patients on haemodialysis. A single oral dose of 250 mg TMP and 200 mg SMP was given to each patient. Multiple samples were collected over 9 days and the following pharmacokinetic parameters were calculated: total area under the plasma level curve, slow disposition rate constant beta and the corresponding t1/2 beta, plasma clearance and the apparent volume of distribution. The results show that the two moieties of the TMP-SMP combination behaved differently in uraemic patients as fas as elimination rate was concerned. TMP was eliminated more slowly both in patients with diminished renal function and in those subjected to haemo- or peritoneal dialysis. The reduction in the rate of elimination of TMP was significantly correlated with the degree of renal impairment. The elimination of SMP, however, was not significantly affected by the reduced renal function; indeed a tendency to increase was noted, at least in dialyzed patients.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗

Prophylactic co-trimoxazole and trimethoprim in the management of urinary tract infection in children.

In a prospective study of low-dose antibacterial prophylaxis of childhood urinary tract infection (UTI), co-trimoxazole and trimethoprim (TMP) have been compared for efficacy in preventing UTI, for their effect on the rectal flora and for secular selection of TMP-resistant organisms. Between 1979 and 1986, 334 children who had proven infection of an unobstructed urinary tract complied in a regimen of low-dose prophylaxis together with measures to eliminate residual urine for at least 6 months. Of these children, 167 had vesico-ureteric reflux and 27 had renal scarring. There was no difference between the two drugs in compliance, which was very good, or in the occurrence of side-effects, which were minimal. Recurrence rates of further infection were 1 per 22 child years for the 226 children receiving cotrimoxazole and 1 per 18 child years for the 108 receiving TMP. All but one of these urinary pathogens were resistant to TMP and reinfection of the urinary tract generally occurred following lapses in attention to complete bladder emptying. Neither a secular increase in recurrent infections during this period, nor a significant change in the proportions of TMP-resistant faecal coliform organisms, was observed. TMP and co-trimoxazole appeared to be equally effective prophylactic agents.

Child↗

Protection by tetramethylpyrazine in acute absolute ethanol-induced gastric lesions.

Acute oral administration of absolute ethanol (1.0 ml/kg) to fasting rats produced extensive necrosis of the gastric mucosa within 1 h. Pretreatment 30 min before administration of ethanol with oral tetramethylpyrazine (TMP) prevented this necrosis. Gross examination of the gastric mucosa of rats that received TMP showed fewer gastric lesions than that of rats who did not receive TMP. TMP pretreatment in rats exhibited superoxide scavenging activity in absolute ethanol-induced lipid peroxidation in gastric mucosal homogenates. TMP added in vitro to gastric homogenates made from control rats also showed scavenging activity. We conclude that the gastric protective mechanism of TMP could be attributed, at least in part, to its ability to inhibit lipid peroxidation and hence indirectly protect the gastric mucosa from oxidative stress.

Animals↗

Protective effect of tetramethylpyrazine on absolute ethanol-induced renal toxicity in mice.

Acute administration of absolute ethanol (10 ml/kg) per os to fasted mice produced extensive renal failure as measured by a rise in blood urea nitrogen and creatinine. Pretreatment with oral administration of tetramethylpyrazine (TMP) prevented such failure. The maximal effect against absolute ethanol-induced renal failure could be observed 1 h after TMP administration. In order to further investigate the renal protective mechanism of TMP, experiments on lipid peroxidation and superoxide scavenging activity were conducted. Renal homogenates made from mice treated with ethanol showed that TMP pretreatment had an antioxidant effect. Mice in acute renal failure had higher malonic dialdehyde concentrations than those pretreated with TMP. The renal protective mechanism of TMP was attributed, in part, to its prominent superoxide scavenging effect, which protects the kidney from superoxide-induced renal damage.

Animals↗

Local cerebral blood flow with prostaglandin E1 or trimethaphan during cerebral aneurysm clip ligation.

This study was performed to examine changes in local cerebral blood flow during hypotensive anaesthesia with either prostaglandin E1 (PGE1) or trimethaphan (TMP). Local cerebral blood flow (LCBF), mean blood pressure (MBP), heart rate (HR), and hourly urine output (UO) were studied in 51 patients undergoing cerebral aneurysm surgery with neuroleptanalgesia (NLA). The incidence of vasospasm after aneurysm surgery, and outcome (Glasgow Outcome Scale) at discharge were evaluated. Measurements of LCBF were made using a thermal gradient blood flow meter. The dose of PGE1 or TMP was adjusted to maintain MBP at about 70 mmHg, and LCBF was studied during and after PGE1 or TMP administration. Hypotensive drugs were discontinued at the completion of aneurysm clipping. After starting PGE1 or TMP, MBP decreased immediately, but HR did not change in either group. The LCBF decreased 30 min after the start of TMP administration and increased immediately after its discontinuation, whereas PGE1 did not affect LCBF. Urine output increased during PGE1 administration but was unchanged during TMP. Neither drug affected surgical outcome or the incidence of vasospasm. These results suggest that PGE1 may be preferable to trimethaphan for hypotensive anaesthesia in cerebral aneurysm surgery because LCBF is maintained.

Adult↗

[Shower PUVA: a new possibility for topical PUVA therapy. Phototoxicity in relation to shower time, water temperature and skin type].

During the past few years, bath PUVA has become established as an effective treatment for various dermatoses and especially for psoriasis. Using 3,4,5 trimethylpsoralen (TMP) in combination with subsequent UVA irradiation, a shower PUVA has been developed as an alternative in local PUVA therapy. This involves moistening the patient's skin - with the exception of the head and neck area - in a shower using water containing psoralen (TMP concentration 0,27 mg/l). The advantages of shower PUVA method are that time, space and cost savings are possible and that only a slight amount of physical exertion is required by the patient standing in the shower compared to immersing the whole body during bath PUVA therapy. The efficacy and practicability of shower PUVA were evaluated using the minimal phototoxic dose (MPD) for healthy volunteers assessing water temperature (33-38 degrees C), shower time (5-10 min), and UVA dose (0,06-1,0 J/cm(2)). Additionally, the time course of TMP-induced photosensitivity was observed over a period of 4 hours after the shower. Using a TMP concentration of 0,27 mg/l, the MPD for skin type I-II lay between 0,125-0,375 J/cm(2) and for skin type III-IV between 0,375-1,0 J/cm(2). Photosensitivity was induced by shower PUVA within 5-10 minutes shower time and at 33-38 degrees C water temperature. MPD exhibited an inverse correlation to temperature but no differences were apparent for shower times between 5 and 10 minutes. Photosensitivity completely disappeared within 2 hours. Minimal phototoxic doses using TMP in shower PUVA are comparable with classical bath PUVA when taking skin type into account. These results support the therapeutic use of shower PUVA using TMP.

Dermatitis, Phototoxic↗

Evidence for acyloxymethyl esters of pyrimidine 5'-deoxyribonucleotides as extracellular sources of active 5'-deoxyribonucleotides in cultured cells.

Cells commonly resist growth inhibition by purine and pyrimidine bases and nucleosides by restricting intracellular formation of the corresponding 5'-mononucleotides. Nucleotide derivatives that can act as effective membrane-transport precursors of the poorly membrane-permeable nucleotides have not been identified so far. We studied the bis(pivaloyloxymethyl)ester (I) of FdUMP (5-fluoro-dUMP) and a cyclic phosphodiester (II) of FdUMP derived from 1,3-dihydroxyl-1-C-(pivaloyloxy-methyl)propane which are active in vivo against a 5-fluoro-2'-deoxyuridine (FUdR)-resistant mouse leukemia and are attacked by carboxylic esterases under physiological conditions to produce FdUMP by elimination of formaldehyde and acrolein respectively. The assay for intracellular FdUMP was the inhibition of DNA synthesis due to inhibition of TMP synthetase in cultured mouse LM(TK-) fibroblasts genetically devoid of thymidine kinase (TK) and thus unable to convert FUdR directly to FdUMP. At 10(-6)M, I, II, or FUdR inhibited DNA synthesis in 2 hr by 99, 80, and 35% respectively; at 10(-5)M. maximal inhibition was attained after less than 15, 30 and 90 min respectively. Inhibition of DNA synthesis in TK+ cells by 10(-5) M I, II, or FUdR was reversed completely by 10(-5)M thymidine (TdR) but unaffected by 10(-5)M UdR, confirming TMP synthetase as the locus of inhibition. At 10(-5)M, bis(pivaloyloxymethyl) esters of phenyl phosphate or a p-substituted benzylphosphonic acid did not inhibit significantly DNA synthesis in TK+ cells. From this finding, and from effects produced by V (see below), we conclude that pivalic acid and CH2O arising from I contribute little to its above inhibitory effects. In TK- cells in which DNA synthesis is prevented by blockade of TMP synthetase with aminopterin, the bis(pivaloyloxymethyl) ester (V) of TMP, at 0.9 x 10(-4) M, induced a 4-fold faster rate of DNA synthesis than did 10(-3)M TMP, whereas 10(-3) M TdR did not affect the rate. After 3 hr the rate with V was 80% that in the absence of aminopterin. In the above systems the nucleotide diesters I, II and V appear to be acting as effective extracellular sources of active intracellular FdUMP and TMP, in processes that involve loss of the two esterifying groups.

Animals↗

Thymidine and zidovudine metabolism in chronically zidovudine-exposed cells in vitro.

Chronic exposure of H9 cells to 25 microM zidovudine (H9-AZT cells) causes a 2- to 3-fold increase in thymidine kinase (TK) activity (Agarwal RP, Int J Purines Pyrimidine Res, in press). The present study compared thymidine (TdR) and AZT anabolism in H9 and H9-AZT cells. After a 3.5-hr incubation with 10 microM TdR or AZT, the total intracellular accumulations of AZT (48.7 microM in H9 cells and 32.8 microM in H9-AZT cells) were 46.4% of TdR accumulation. Other major differences between TdR and AZT anabolism were: (i) the majority of TdR (84-87%) was incorporated into DNA compared to less than 1% of AZT; and (ii) whereas distribution of TdR in the nucleotides was TTP greater than TMP greater than TDP, zidovudine distributed was AZT-MP much greater than AZT-TP much greater than AZT-DP. Because of the poor substrate activity of AZT-MP for thymidylate kinase (TMP-kinase), most of the AZT (95-98%) remained as AZT-MP. TMP-kinase activities with TMP as substrate were 47.6 +/- 20.3 and 91.4 +/- 28.8 pmol/mg protein/min in H9 and H9-AZT cells, respectively. 5'-Nucleotidase activities with TMP as substrate were 428.9 +/- 37.8 and 255.9 +/- 28.7 pmol/mg protein/min in H9 and H9-AZT cells, respectively. Activities of these enzymes with AZT-MP as a substrate were very low. Despite an increase in TK and TMP-kinase, and a decrease in 5'-nucleotidase activities, the total intracellular accumulations of TdR and AZT were reduced significantly (P less than 0.05) to 67.5% in H9-AZT cells. Thymidine transport (0.66 to 0.68 pmol/sec/10(6) cells) was similar in both the cell lines. The severe reductions of TdR salvage caused by chronic exposure of cells to AZT, if it occurs in AIDS patients on AZT chemotherapy, may explain some of the long-term clinical toxicities of the drug.

Cells, Cultured↗

Baquiloprim, a new antifolate antibacterial: in vitro activity and pharmacokinetic properties in cattle.

During examination of the half-lives in cattle of a series of 5-substituted diaminobenzyl-pyrimidines, it was found that replacement of the phenyl ring of trimethoprim (TMP) by bicyclic structures, particularly a quinolyl group, led to increases in half-life. The presence of a dimethylamino group on the quinolyl ring of the compound baquiloprim (BQP) conferred a half-life of about 10 hours. In contrast to TMP (half-life about one hour), BQP was well absorbed from the gastrointestinal tract in all ages of cattle, plasma concentrations reaching a plateau on the day after dosing followed by a slow decline. BQP showed the same high broad spectrum antibacterial activity as TMP, with marked synergy with sulphonamides. Its differential binding of the dihydrofolate reductases of Escherichia coli and rat liver predicted a margin of safety similar to that of TMP. The results of efficacy studies in mice in comparison with TMP showed that the longer half-life of BQP was associated with greater efficacy, and therapeutic properties superior to those of TMP in cattle were therefore predicted for BQP.

Administration, Oral↗

Role of metabolic activation, covalent binding, and glutathione depletion in pulmonary toxicity produced by an impurity of malathion.

O,O,S-Trimethyl phosphorothioate (OOS-TMP) is an impurity present in widely used organophosphorus insecticides such as malathion. Oral treatment of rats with the compound produces prominent bronchiolar epithelial necrosis. Following the administration of [3H]OOS-TMP to rats, substantial amounts of radiolabeled material were covalently bound to lung with a concomitant depletion of glutathione (GSH). Other organs showing significant covalently bound radioactivity were liver, kidneys, and ileum. The maximal accumulation occurred in the tissues within 6 hr, and reached a plateau between 6 and 12 hr. Pretreatment of rats with either phenobarbital or piperonyl butoxide decreased the level of radiolabeled material bound in lung, GSH depletion, and the toxicity of OOS-TMP. These results suggest that the covalent binding is due to a metabolite(s) of OOS-TMP and that the metabolite(s) is involved in the mechanism of toxicity of OOS-TMP. Pulmonary GSH may play a protective role against OOS-TMP induced lung toxicity.

Administration, Oral↗

Cellular responses to O,O,S-trimethyl phosphorothioate-induced pulmonary injury in rats.

O,O,S-Trimethyl phosphorothioate (OOS-TMP), an impurity of many organophosphorus insecticides, causes a delayed toxicity in rats and mice which is associated with morphological and biochemical changes in the lung. Oral administration of doses as low as 20 mg/kg alters bronchiolar epithelial morphology and causes an increase in bronchopulmonary lavage lactate dehydrogenase levels. In the present study, the effects of OOS-TMP on alveolar and bronchiolar cells were examined by determining the patterns of cellular regeneration in rats at periods of 12 hr, 24 hr, 3 days, and 7 days after treatment. Dividing cells were labeled with tritiated thymidine and studied with autoradiographic techniques. The results showed that OOS-TMP treatment initiated proliferation of alveolar type II cells within 24 hr. The proliferative response of type II cells continued to increase in 3-day and 7-day treatment groups. Labeled alveolar type I cells began to appear after 3 days, indicating that type II cells were dividing to replace damaged type I cells. Cells of the alveoli were thickened and showed vacuolization. In the bronchioles, labeled Clara cells were increased on Day 3 and Day 7 while the number of labeled ciliated cells remained near control levels throughout all time points, indicating that in bronchiolar epithelium, OOS-TMP stimulates the proliferation of Clara cells but does not damage ciliated cells. The binding of tritiated OOS-TMP to lung tissue was also examined by autoradiography. It was found that [3H]OOS-TMP binds to all regions of lung tissue.

Animals↗

Immunosuppressive effect of an impurity of malathion: inhibition of murine T- and B-lymphocyte responses by O,O,S-trimethyl phosphorothioate.

The effect of O,O,S-trimethyl phosphorothioate (OOS-TMP) on immune responses such as antigen presentation, antibody production, and cytotoxic T-lymphocyte (CTL) function was examined in vitro. The roles of non-enzymatic and enzymatic glutathione (GSH) conjugation of OOS-TMP in these responses were studied. Antibody responses to T-dependent and T-independent antigens were evaluated after (i) direct culture with spleen or B cells; (ii) cocultivation of B cells with T cells with and without preincubation of OOS-TMP with GSH fortified cytosol. Antigen presentation by macrophages was also assessed after such treatment as compared to untreated controls. OOS-TMP preincubated with GSH had an inhibitory effect on the cytotoxic T lymphocyte and the direct hemolytic plaque forming cell responses. This was found to be mediated by a direct inhibitory effect on macrophages, T, and B cells of the immune system and not through the generation of regulatory suppressor T cells. Thus, the mode of suppressive action of OOS-TMP in vitro is due to inhibition of lymphocytic proliferation. This is only possible in the presence of glutathione which was determined to be a prerequisite for the induction of OOS-TMP suppressive effect.

Animals↗

Analysis of a human immunodeficiency virus type 1 isolate carrying a truncated transmembrane glycoprotein.

We have recently reported the isolation of a human immunodeficiency virus type 1 (HIV-1), KB-1gp32 carrying a shorter size (32 kDa) of transmembrane glycoprotein (TMP) from TALL-1 cells persistently infected with KB-1gp41 virus strain (Shimizu et al., 1990a). Endoglycosidase treatments showed that the different size of the TMP between the two strains was due to a truncation of 9 kDa of polypeptide in the KB-1gp32 TMP coding region. Sequence analysis revealed the substitution of a CAG codon to a TAG stop codon just downstream of the putative membrane-spanning domain of the TMP of KB-1gp32. This resulted in a truncation of some 133 amino acids of the cytoplasmic domain of TMP. The data indicate that a premature stop codon in KB-1gp32 has been introduced during adaption of the parental virus to TALL-1 cells. We have constructed two chimeric clones between the env region of a clone pKB-1, derived from KB-1gp32, and an infectious molecular clone pNL-432. We have also constructed a site-directed mutant of pNL-432 carrying a premature stop codon at the same position as the env stop codon of pKB-1. Among the three clones carrying a premature stop codon in env, only one chimeric clone was infectious to TALL-1 but not MT-2 cells. This clone contained the entire tat, rev, vpu, and env genes of pKB-1. The pNL-432 mutant was not infectious. The results suggest that some sequences of pKB-1 might compensate for the truncation of the TMP during replication in TALL-1 cells.

Amino Acid Sequence↗

Substrate-inhibitor cooperative interactions with microbial dihydrofolate reductases.

Cooperativity in the binding of two substrates to an enzyme is a now well-established phenomenon. The x-ray crystallographic structure of the E. coli DHFR binary TMP complex compared with the ternary enzyme-NADPH-TMP complex suggests without too imaginative extrapolation, that the conformational changes resulting from the binding of one ligand aid in favorably positioning potential binding sites for the second ligand. Of greater importance is the fact that the extent to which inhibitor binding is enhanced by the binding of NADPH varies from species to species. To a significant extent, for example, the selectivity of TMP is enhanced by the increase in its binding to the E. coli enzyme when NADPH is present as compared with several mammalian enzymes. The reverse, negative cooperativity (a decrease in binding of a substance when moving from the binary to a ternary complex), is perhaps less common and certainly less well studied. The present paper deals with one such enzyme, the DHFR from C. albicans, and by reference to another, that from S. cerevisiae, where it is shown that the binding of substrates exhibit strong negative cooperativity. It was of interest also to determine the relationship between inhibitor/NADPH cooperativity and the relative insensitivity of N. gonorrhoeae to TMP. Equilibrium studies show that the binding of TMP in binary complex with this enzyme is exceedingly poor and that a 2,200-fold cooperative effect brings the gonococcal enzyme Ki within one order of magnitude of the E. coli enzyme Ki. Even so, it takes synergism of another sort (with sulfamethoxazole) and high doses to make co-trimoxazole therapy feasible for treating gonorrhoeae. The comparative results on the gonococcal enzyme for a family of near relatives of TMP are of interest also for the reason that the structure-activity relationships with this enzyme are quite different from those of the E. coli and other microbial enzymes. Finally, it should be pointed out that although the negative cooperativity found for the candida and saccharomyces enzymes is relatively large, it is the values of the substrate Michaelis constants that are physiologically relevant. The Km values of the yeast enzymes are within the range for other DHFR and therefore the intracellular activity of the enzymes should not be compromised.

Candida albicans↗