A field test of the use of pop-off data storage tags in freshwater fishes.

In the present study, pop-off data storage tags (pDST) without any transmitting capabilities were attached to 118 adult salmonids in a 19 000 km2 freshwater system (Lake Ontario). The 9·3 cm long cylindrical tags were externally attached to fishes using a backpack-style harness, set to record pressure (dBar ≈ depth in m) and temperature every 70 s (and at some key times, every 5 s) and programmed to release from the harness and float to the surface after c. 1 year. Recapture of the bright-orange tags for data retrieval relied on members of the public finding tags on shore, or on anglers capturing fishes with tags attached and using the contact information displayed on each tag to mail tags to the research team in exchange for a monetary reward. Thirty-seven tags were found and returned from the 118 released (31%), while 26 of the 118 tags (22%) remained scheduled to pop-off in summer 2017. Of the 37 tags returned, 23 were from wild-caught fishes (out of 88 wild-caught and tagged fishes; 26%) and yielded useful data whereas 14 were from hatchery-reared fishes that were opportunistically tagged and appear to have been unable to acclimate to life in the wild and died days to weeks after release. The field study described here thus demonstrated that pDSTs can be a viable option for collecting large amounts of high-resolution depth and temperature data for salmonids in freshwater systems. Technical challenges, limitations and unknowns related to the use of pDSTs with freshwater fishes are discussed. In addition, pDSTs are compared with alternate electronic tagging technologies and assessed for their potential as a more widespread tool in research on freshwater fishes.

Tissue-specific turnover and diet-tissue discrimination factors of carbon and nitrogen isotopes of a common forage fish held at two temperatures.

RATIONALE: The application of stable isotopes to foraging ecology is dependent on understanding life-history and environmental factors unrelated to diet that may influence isotopic composition. Diet-tissue discrimination factors (DTDFs) and turnover rates will increase the accuracy of isotope-based studies. Furthermore, little consideration has been given to the effects of temperature or life-history stage on isotopic ratios despite the prevalence of variation in temperature and growth rates throughout life. METHODS: We measured δ13 C and δ15 N values with an elemental analyzer coupled to a continuous flow isotope ratio mass spectrometer. These values were used to estimate turnover and DTDFs for Emerald Shiners (Notropis atherinoides), a common North American freshwater forage fish. Fish were assigned to a temperature treatment, either 10°C (Low) or 20°C (High), and provided one of three diets (commercial pellet, Artemia salina, or Hemimysis anomala). At regular intervals fish were sampled and the isotopic compositions of whole body and liver tissues were determined. RESULTS: Tissue turnover rates for fish fed Artemia were faster for liver than for whole body, but were also influenced by temperature. Turnover occurred faster at higher temperatures for body and liver δ15 N values, but not for δ13 C values. The pellet and Hemimysis treatments were in isotopic equilibrium from the start of the experiment and estimated DTDFs based on these treatments were lower than assumed for Δ15 N (+0.6 to 2.7‰) and variable, but within expected ranges for Δ13 C (-1.9 to +1.5‰). CONCLUSIONS: The results for Emerald Shiners differed from commonly made assumptions for applying stable isotopes to ecological questions, possibly related to a bias in the use of juveniles in studies of turnover and DTDFs and assumptions regarding thermal-independence of isotopic relationships. The species-specific DTDF and tissue turnover estimates provided here will inform interpretations of stable isotope data for smaller fish species and improve food-web studies.

Clathrin localization and dynamics in Aspergillus nidulans.

Cell growth necessitates extensive membrane remodeling events including vesicle fusion or fission, processes that are regulated by coat proteins. The hyphal cells of filamentous fungi concentrate both exocytosis and endocytosis at the apex. This investigation focuses on clathrin in Aspergillus nidulans, with the aim of understanding its role in membrane remodeling in growing hyphae. We examined clathrin heavy chain (ClaH-GFP) which localized to three distinct subcellular structures: late Golgi (trans-Golgi equivalents of filamentous fungi), which are concentrated just behind the hyphal tip but are intermittently present throughout all hyphal cells; the region of concentrated endocytosis just behind the hyphal apex (the "endocytic collar"); and small, rapidly moving puncta that were seen trafficking long distances in nearly all hyphal compartments. ClaH localized to distinct domains on late Golgi, and these clathrin "hubs" dispersed in synchrony after the late Golgi marker PHOSBP . Although clathrin was essential for growth, ClaH did not colocalize well with the endocytic patch marker fimbrin. Tests of FM4-64 internalization and repression of ClaH corroborated the observation that clathrin does not play an important role in endocytosis in A. nidulans. A minor portion of ClaH puncta exhibited bidirectional movement, likely along microtubules, but were generally distinct from early endosomes.

Utilization of stomach content DNA to determine diet diversity in piscivorous fishes.

The objective of the study was to validate and apply DNA-based approaches to describe fish diets. Laboratory experiments were performed to determine the number of hours after ingestion that DNA could be reliably isolated from stomach content residues, particularly with small prey fishes (c. 1 cm, <0·75 g). Additionally, experiments were conducted at different temperatures to resolve temperature effects on digestion rate and DNA viability. The molecular protocol of cloning and sequencing was then applied to the analysis of stomach contents of wild fishes collected from the western basin of Lake Erie, Canada-U.S.A. The results showed that molecular techniques were more precise than traditional visual inspection and could provide insight into diet preferences for even highly digested prey that have lost all physical characteristics.

GABA(A) receptors containing (alpha)5 subunits in the CA1 and CA3 hippocampal fields regulate ethanol-motivated behaviors: an extended ethanol reward circuitry.

GABA receptors within the mesolimbic circuitry have been proposed to play a role in regulating alcohol-seeking behaviors in the alcohol-preferring (P) rat. However, the precise GABA(A) receptor subunit(s) mediating the reinforcing properties of EtOH remains unknown. We examined the capacity of intrahippocampal infusions of an alpha5 subunit-selective ( approximately 75-fold) benzodiazepine (BDZ) inverse agonist [i.e., RY 023 (RY) (tert-butyl 8-(trimethylsilyl) acetylene-5,6-dihydro-5-methyl-6-oxo-4H-imidazo [1,5a] [1,4] benzodiazepine-3-carboxylate)] to alter lever pressing maintained by concurrent presentation of EtOH (10% v/v) and a saccharin solution (0.05% w/v). Bilateral (1.5-20 microgram) and unilateral (0.01-40 microgram) RY dose-dependently reduced EtOH-maintained responding, with saccharin-maintained responding being reduced only with the highest doses (e.g., 20 and 40 microgram). The competitive BDZ antagonist ZK 93426 (ZK) (7 microgram) reversed the RY-induced suppression on EtOH-maintained responding, confirming that the effect was mediated via the BDZ site on the GABA(A) receptor complex. Intrahippocampal modulation of the EtOH-maintained responding was site-specific; no antagonism by RY after intra-accumbens [nucleus accumbens (NACC)] and intraventral tegmental [ventral tegmental area (VTA)] infusions was observed. Because the VTA and NACC contain very high densities of alpha1 and alpha2 subunits, respectively, we determined whether RY exhibited a "negative" or "neutral" pharmacological profile at recombinant alpha1beta3gamma2, alpha2beta3gamma2, and alpha5beta3gamma2 receptors expressed in Xenopus oocytes. RY produced "classic" inverse agonism at all alpha receptor subtypes; thus, a neutral efficacy was not sufficient to explain the failure of RY to alter EtOH responding in the NACC or VTA. The results provide the first demonstration that the alpha5-containing GABA(A) receptors in the hippocampus play an important role in regulating EtOH-seeking behaviors.

Serotonergic neuronal activity in the dorsal raphe nucleus of selectively bred alcohol-preferring and alcohol-nonpreferring rats and unselected wistar rats.

BACKGROUND: Selectively-bred alcohol-preferring (P) rats have fewer serotonin (5-HT) neurons in the dorsal raphe nucleus (DRN) than do alcohol-nonpreferring (NP) rats. The present study was designed to test the hypothesis that the remaining 5-HT neurons in P rats compensated for their reduced number by increasing neuronal activity. METHODS: Spontaneous activity was recorded from single-spiking and bursting 5-HT neurons in the DRN of unanesthetized paralyzed, alcohol-naive P, NP, and Wistar rats. Firing frequencies, the percentages of action potentials in bursts, and the percentages of bursting neurons were evaluated. RESULTS: There were no significant differences among the three groups of rats in any of the parameters measured. Power analyses were performed on preliminary data to determine the sample sizes necessary for detection of significant differences. The mean firing frequencies of single-spiking 5-HT neurons averaged 1.8 (37 neurons), 1.7 (17 neurons), and 1.8 (41 neurons) spikes per second in P, NP, and Wistar rats, respectively. For bursting 5-HT neurons, the percentages of action potentials in bursts for P, NP, and Wistar rats were 55.0% (24 neurons), 49.7% (18 neurons), and 55.1% (21 neurons). The mean percentages of bursting 5-HT neurons encountered per electrode penetration were 44% for P rats (n = 28), 44% for NP rats (n = 14), and 34% for Wistar rats (n = 26). CONCLUSIONS: The results indicate that the sample of 5-HT neurons recorded in the DRN of P rats had not compensated for a reduced number by altering neuronal activity.

Neonatal binge ethanol exposure using intubation: timing and dose effects on place learning.

Neonatal rats were given ethanol using an acute intubation procedure that resulted in daily binge-like exposure with minimal effects on somatic growth. Acquisition of place learning in the Morris water maze was evaluated on postnatal days (PD) 26-31. In Experiment 1, a total of 5.25 g/kg/day of ethanol was administered in two daily intubations on PD 4-6, PD 7-9, or PD 4-9, producing mean peak BACs of 265 mg/dL. Place learning acquisition deficits in a 114-cm-diameter tank were found for the PD 4-9 and PD 7-9 groups, but not the PD 4-6 group. In Experiment 2, either 4.5 or 5.25 g/kg/day of ethanol was administered on PD 7-9 and place learning was tested in a 171-cm-diameter tank. Significant acquisition deficits resulted from the higher dose, and probe trial search patterns for both ethanol groups were significantly less localized than controls. In Experiment 3, no significant effects of either PD 7-9 dose were found on a visible platform task. These findings reveal selective place learning deficits in this intubation model of neonatal binge exposure, and confirm a temporal window of vulnerability to spatial learning deficits during the second neonatal week.

More real-time visual and haptic interaction with anatomical data.

The University of Colorado's Center for Human Simulation has developed fundamental algorithms for real-time visual and haptic interaction with polygonal and voxel base data sets, including those derived from the Visible Human Dataset. These algorithms are currently being used to create prototype simulators for surgery, needle insertion (anesthesiology, radiology, and rheumatology), dentistry, and ophthalmology. This paper briefly discusses our segmentation and classification effort as well as our ability to create texture-mapped polygonal models directly from the data: both are fundamental to the creation of anatomically based simulators.

Lack of proarrhythmia as assessed by Holter monitor after atrial radiofrequency ablation of supraventricular tachycardia in children.

The purpose of this study was to assess the short-term arrhythmogenicity of atrial radiofrequency (RF) ablation lesions in children. Patients with the greatest exposure to RF energy comprised the study group. Holter data on 35 RF ablation procedures in 31 patients with a median age of 13.2 years (range 3 months to 20 years) was retrospectively analyzed. Patients received an average of 19.9 (SD = 13.6) RF lesions, all delivered by an atrial approach. Supraventricular ectopy and ventricular ectopy were compared immediately before and after and 4 to 9 weeks after RF ablation by serial Holter monitoring. Factors thought to possibly predispose patients to a proarrhythmic effect were used to define subgroups for separate analysis. No increase in ambient supraventricular ectopy or ventricular ectopy was observed either immediately after or 4 to 9 weeks after RF ablation compared with the baseline Holter recordings. Children exposed to relatively large doses of RF energy may demonstrate transient and asymptomatic nonsustained tachycardias in the short term. However, no new sustained tachycardias and no increase in supraventricular or ventricular ambient ectopy are detected by short-term Holter monitoring.

Management of atrial flutter after the Fontan procedure.

OBJECTIVES: The purpose of this study was to review the management of atrial flutter occurring after the Fontan procedure. BACKGROUND: Atrial flutter occurs frequently after the Fontan procedure and is often hemodynamically poorly tolerated. METHODS: The patients' charts were reviewed for relevant information. RESULTS: Between 1984 and 1992, 18 patients had atrial flutter after the Fontan procedure. The underlying heart defect was tricuspid atresia in nine, mitral atresia in six and double inlet left ventricle in three. All but three patients had undergone previous palliative surgery. The time interval from Fontan operation to atrial flutter was < 1 day to 16 years (mean 3.7 years). Seven had early atrial flutter before leaving the hospital. Electrophysiologic study in 15 showed sinus node dysfunction in 12. Atrial flutter was inducible in all patients, and 13 had > 1 flutter configuration. Digoxin and a variety of other antiarrhythmic agents (mean 2.7 drugs/patient) were tried with poor results. Only digoxin, amiodarone, flecainide and propafenone showed some benefit when used alone or in combination. Antitachycardia pacemakers were implanted in 16 patients (endocardial 14, epicardial 2) and, with drugs, were useful in 8 (50%). Because atrial flutter was resistant to treatment, right atriectomy was performed in three patients (with benefit in two, one death), successful radiofrequency catheter His bundle ablation in one patient and catheter ablation of atrial flutter in three patients (two failed, one partial success). One patient underwent heart transplantation, and two died suddenly. Another died of complications after an elective epicardial pacemaker replacement procedure. CONCLUSIONS: Atrial flutter after the Fontan procedure is difficult to control. Aggressive drug and antitachycardia pacemaker therapy help about half of the patients. When these measures fail, other options, such as atriectomy, His bundle ablation or catheter ablation of atrial flutter, need consideration. The risk of sudden death justifies the use of such aggressive treatment methods.

Electrical stimulation of contractile activity accelerates growth of cultured neonatal cardiocytes.

An electrical stimulation system was designed to regulate synchronized contractile activity of neonatal rat cardiocytes and to examine the effects of mechanical contraction on cardiocyte growth. Continuous electrical stimulation at a pulse duration of 5 milliseconds and frequency of 3 Hz resulted in a time-dependent accumulation of cell protein that reached 34% above initial values, as measured by the protein-to-DNA ratio. The growth response did not occur using voltage amplitudes that were subthreshold for contraction and was independent of contraction frequencies set at > or = 0.5 Hz. The RNA-to-DNA ratio increased in parallel to cell protein, indicating that the capacity for protein synthesis was enhanced by contraction. Rates of 28S rRNA synthesis were accelerated twofold in contracting cardiocytes. By comparison, protein and RNA accumulation did not occur in electrically stimulated cardiocytes in which contraction was blocked by either 10 mumol/L verapamil or by 5 mmol/L 2,3-butanedione monoxime, an inhibitor of actomyosin crossbridge cycling. Electrical stimulation of cardiocyte contraction did not enhance alpha-cardiac actin or myosin heavy chain (alpha+beta) mRNA transcript levels relative to 28S rRNA during the period of rapid growth that occurred over the first 48 hours. It is concluded that (1) electrical stimulation of contraction accelerates cardiocyte growth and RNA accumulation, (2) mechanical contraction is involved in regulating the growth of electrically stimulated cardiocytes, and (3) the levels of alpha-actin and myosin heavy chain mRNA increase in proportion to rRNA during the growth of contracting cardiocytes.

Echocardiographic and anatomic correlation of ventricular septal defect morphology in newborn Yucatan pigs.

With the use of a Yucatan micropig strain with a high incidence of ventricular septal defects (VSDs), results of two-dimensional and color-flow Doppler echocardiography of VSD morphology in newborn piglets were correlated with autopsy findings. A spectrum of perimembranous, muscular outlet, and doubly committed subarterial VSDs was found. Echocardiography was performed in 29 piglets weighing 1.2 to 4.4 (mean 2.8) kg, studied at age 4 to 18 (mean 8) days. VSD was diagnosed by means of echocardiography in 16 of 29 subjects; morphologic findings included perimembranous defects in 12, muscular outlet in two, and doubly committed subarterial defect in two. At autopsy the presence and location of defects were confirmed in all pigs. No additional defects were found. VSD diameters were 1.0 to 5.0 (mean 3.94) mm on echocardiography and 1.0 to 6.0 (mean 2.84) mm at autopsy. After aortic valve diameter was used as an internal control for tissue shrinkage during fixation, echocardiography/color Doppler imaging tended to overestimate VSD diameter by 21% (0.6 mm). In conclusion, echocardiography/Doppler imaging accurately identified the presence, morphology, and size of even the smallest VSDs in newborn Yucatan micropigs. Echocardiographic classification of VSD morphology in vivo will facilitate future research on specific types of VSDs in this animal model.

Characterization of two genes encoding Bacillus thuringiensis insecticidal crystal proteins toxic to Coleoptera species.

Bacillus thuringiensis EG2838 and EG4961 are highly toxic to Colorado potato beetle larvae, and only strain EG4961 is toxic to southern corn rootworm larvae. To investigate the cause of the different insecticidal activities of EG2838 and EG4961, cryIII-type genes toxic to coleopterans were cloned from each strain. The cryIIIB gene, cloned as part of an 8.0-kb EcoRI fragment of EG2838 DNA, encoded a crystal protein (CryIIIB) of 74,237 Da. The cryIIIB2 gene, cloned as part of an 8.3-kb PstI-Asp718 fragment of EG4961 DNA, encoded a crystal protein (CryIIIB2) of 74,393 Da that was 94% identical to CryIIIB. Analysis of the transcriptional start sites showed that cryIIIB and cryIIIB2 were initiated from a conserved region located within 130 nucleotides upstream from the translation start sites of both genes. Although the CryIIIB and CryIIIB2 proteins were similar in sequence, they displayed distinct insecticidal activities: CryIIIB was one-third as toxic as CryIIIB2 to Colorado potato beetle larvae, and CryIIIB2, but not CryIIIB, was toxic to southern corn rootworm larvae. Genes encoding crystal proteins of approximately 32 and 31 kDa were located adjacent to the cryIIIB and cryIIIB2 genes, respectively. The 32- and 31-kDa crystal proteins failed to enhance the insecticidal activities of CryIIIB and CryIIIB2.

Two novel strains of Bacillus thuringiensis toxic to coleopterans.

Two novel strains of Bacillus thuringiensis were isolated from native habitats by the use of genes coding for proteins toxic to coleopterans (cryIII genes) as hybridization probes. Strain EG2838 (isolated by the use of the cryIIIA probe) contained a cryIIIA-hybridizing plasmid of approximately 100 MDa and synthesized crystal proteins of approximately 200 (doublet), 74, 70, 32, and 28 kDa. Strain EG4961 (isolated by the use of a cryIIIA-related probe) contained a cryIIIA-hybridizing plasmid of approximately 95 MDa and synthesized crystal proteins of 74, 70, and 30 kDa. Structural relationships among the crystal proteins of strains EG2838 and EG4961 were detected; antibodies to the CryIIIA protein toxic to coleopterans reacted with the 74- and 70-kDa proteins of EG2838 and EG4961, antibodies to the 32-kDa plus 28-kDa proteins of EG2838 reacted with the 30-kDa protein of EG4961, and antibodies to the 200-kDa proteins of EG2838 reacted with the 28-kDa protein of EG2838. Experiments with B. thuringiensis flagella antibody reagents demonstrated that EG2838 belongs to H serotype 9 (reference strain B. thuringiensis subsp. tolworthi) and that EG4961 belongs to H serotype 18 (reference strain B. thuringiensis subsp. kumamotoensis). A mixture of spores plus crystal proteins of either EG2838 or EG4961 was toxic to the larvae of Colorado potato beetle (Leptinotarsa decemlineata), and significantly, the EG4961 mixture was also toxic to the larvae of southern corn rootworm (Diabrotica undecimpunctata howardi). DNA restriction blot analysis suggested that strains EG2838 and EG4961 each contained a unique gene coding for a protein toxic to coleopterans.

Isolation and characterization of a novel insecticidal crystal protein gene from Bacillus thuringiensis subsp. aizawai.

Bacillus thuringiensis subsp. aizawai EG6346, a novel grain dust isolate, was analyzed by Southern blot hybridization for its insecticidal crystal protein (ICP) gene profile. Strain EG6346 lacks previously characterized cryIA ICP genes yet does possess novel cryI-related gene sequences. A recombinant genomic plasmid library was constructed for strain EG6346 in Escherichia coli. One recombinant plasmid, pEG640, isolated from the library contained a novel ICP gene on a 5.7-kb Sau3A insert. The sequence of this gene, designated cryIF, was related to, but distinct from, the published sequences for other cryI genes. A second novel cryI-related sequence was also located on pEG640, approximately 500 bp downstream from cryIF. Introduction of cryIF into a Cry- B. thuringiensis recipient strain via electroporation enabled sufficient production of CryIF protein for quantitative bioassay analyses of insecticidal specificity. The CryIF crystal protein was selectively toxic to a subset of lepidopteran insects tested, including the larvae of Ostrinia nubilalis and Spodoptera exigua.

Insecticidal toxins from Bacillus thuringiensis subsp. kenyae: gene cloning and characterization and comparison with B. thuringiensis subsp. kurstaki CryIA(c) toxins.

Genes encoding insecticidal crystal proteins were cloned from three strains of Bacillus thuringiensis subsp. kenyae and two strains of B. thuringiensis subsp. kurstaki. Characterization of the B. thuringiensis subsp. kenyae toxin genes showed that they are most closely related to cryIA(c) from B. thuringiensis subsp. kurstaki. The cloned genes were introduced into Bacillus host strains, and the spectra of insecticidal activities of each Cry protein were determined for six pest lepidopteran insects. CryIA(c) proteins from B. thuringiensis subsp. kenyae are as active as CryIA(c) proteins from B. thuringiensis subsp. kurstaki against Trichoplusia ni, Lymantria dispar, Heliothis zea, and H. virescens but are significantly less active against Plutella xylostella and, in some cases, Ostrinia nubilalis. The sequence of a cryIA(c) gene from B. thuringiensis subsp. kenyae was determined (GenBank M35524) and compared with that of cryIA(c) from B. thuringiensis subsp. kurstaki. The two genes are more than 99% identical and show seven amino acid differences among the predicted sequences of 1,177 amino acids.

The role of cellular folates in the enhancement of activity of the thymidylate synthase inhibitor 10-propargyl-5,8-dideazafolate against hepatoma cells in vitro by inhibitors of dihydrofolate reductase.

Exposure of growing cultures of hepatoma cells in vitro to the lipid-soluble dihydrofolate reductase inhibitors metoprine (36 nM) or trimetrexate (2 nM) at subtoxic concentrations causes little change in cell growth rate, colony forming ability, cell cycle distribution, and de novo purine and thymidylate biosynthesis. The reductase inhibitors augment the cytotoxic activity of the thymidylate synthase inhibitor, 10-propargyl-5,8-dideazafolate by nearly 10-fold under optimal conditions. Treatment of the hepatoma cells with the reductase inhibitors for 72 h during growth caused approximately a 75% reduction in total cellular folates and 5,10-methylenetetrahydrofolate (primarily as polyglutamates) the substrate for thymidylate synthase. The reductase inhibitors also cause a doubling in the accumulation of 10-propargyl-5,8-dideazafolate polyglutamates. The combined antifolate treatment (metoprine or trimetrexate plus 10-propargyl-5,8-dideazafolate) expands the dUMP pool by 30-fold, which is more than the sum of either of the antifolates alone. Consequently, it is postulated that the enhanced activity of 10-propargyl-5,8-dideazafolate in combination with low concentrations of dihydrofolate reductase inhibitors is due to an increase in the ratio of inhibitor to substrate for thymidylate synthase of nearly 10-fold and an extensive enhancement of the dUMP pool. These conditions predispose the target enzyme and the cells to more effective metabolic blockade by 10-propargyl-5,8-dideazafolate which is presumably caused by the formation of an inhibited 10-propargyl-5,8-dideazafolate[polyglutamate]-thymidylate synthase-dUMP ternary complex.

The effect of methionine on methotrexate metabolism in rat hepatocytes in monolayer culture.

The effect of methyl donors on the metabolism of methotrexate has been investigated in rat hepatocytes in monolayer culture. Pulse exposure to low concentrations of methotrexate (1 microM, 3h) in the absence of methionine results in the facile formation of the di- to pentaglutamates with the di- and triglutamate predominating. Further incubation after the removal of methotrexate (MTX) results in a shift to the tetra- and pentaglutamate at the expense of the shorter chain length derivatives. The same measurement in the presence of 1 mM methionine causes approx. an 80% inhibition in the formation of polyglutamates. This effect can be partially achieved when methionine is replaced by choline or betaine. No alteration in the formation of 7-hydroxymethotrexate could be detected by similar changes in methionine concentrations in the medium. The activity of the enzymes which synthesize and degrade methotrexate polyglutamates, folylpolyglutamate synthetase and gamma-glutamyl hydrolase, respectively, were the same in extracts of cells grown in the absence and in the presence of 1 mM methionine. Incubation of the hepatocytes with methionine causes a significant increase in 5,6,7,8-tetrahydrofolate (H4folate), 5,10-methylenehydrofolate and 10-formyltetrahydrofolate and a decrease in 5-methyltetrahydrofolate. These results suggest that the inhibition of glutamylation of methotrexate could be due in part to an elevation in reduced folates which can more effectively compete with methotrexate as a substrate for folylpolyglutamate synthetase. Inhibition in methotrexate glutamylation by methionine, betaine and choline in hepatocytes may contribute to the alleviation of hepatic toxicity by methyl donors.

The enhancement of the activity of 10-propargyl-5,8-dideazafolate and 5,10-dideazatetrahydrofolate by inhibitors of dihydrofolate reductase.

Treatment of H35 hepatoma cells with the lipid soluble dihydrofolate reductase inhibitors metoprine and trimetrexate cause a nearly 10-fold increase in the toxicity of the antipyrimidine folate analogue PDDF and the antipurine folate analogue DDATHF. Evaluation of these interactions by the combination index developed by Chou (17-20) yields results conforming to synergistic interactions. The capacity of PDDF to inhibit thymidylate synthase in intact cells as measured by tritium release from [5-3H]deoxyuridine was increased by approximately the same amount by preincubation with the dihydrofolate reductase inhibitors. The primary effect of the reductase inhibitors in causing greater activity may be a reduction in cellular folates which can cause 5,10-CH2H4PteGlun to decrease and cellular PDDF (polyglutamates) to increase. These conditions would favor inhibition of thymidylate synthase by PDDF by promoting formation of the stable, inhibited PDDF (polyglutamates)-thymidylate synthase-dUMP complex (12).