Mutation of a glutamate receptor motif reveals its role in gating and delta2 receptor channel properties.

Despite its importance in the cerebellum, the functions of the orphan glutamate receptor delta2 are unknown. We examined a mutant delta2 receptor channel in lurcher mice that was constitutively active in the absence of ligand. Because this mutation was within a highly conserved motif (YTANLAAF), we tested its effect on several glutamate receptors. Mutant delta2 receptors showed distinct channel properties, including double rectification of the current-voltage relationship, sensitivity to a polyamine antagonist and moderate Ca 2+ permeability, whereas other constitutively active mutant glutamate channels resembled wild-type channels in these respects. Moreover, the kinetics of ligand-activated currents were strikingly altered. We conclude that the delta2 receptor has a functional ion channel pore similar to that of glutamate receptors. The motif may have a role in the channel gating of glutamate receptors.

Glucocorticoid receptor activation is involved in producing abnormal phenotypes of single-prolonged stress rats: a putative post-traumatic stress disorder model.

Post-traumatic stress disorder (PTSD) is a stress-related mental disorder caused by traumatic experience, and presents with characteristic symptoms, such as intrusive memories, a state of hyperarousal, and avoidance, that endure for years. Single-prolonged stress (SPS) is one of the animal models proposed for PTSD. Rats exposed to SPS showed enhanced inhibition of the hypothalamo-pituitary-adrenal (HPA) axis, which has been reliably reproduced in patients with PTSD, and increased expression of glucocorticoid receptor (GR) in the hippocampus. In this study, we characterized further neuroendocrinologic, behavioral and electrophysiological alterations in SPS rats. Plasma corticosterone recovered from an initial increase within a week, and gross histological changes and neuronal cell death were not observed in the hippocampus of the SPS rats. Behavioral analyses revealed that the SPS rats presented enhanced acoustic startle and impaired spatial memory that paralleled the deficits in hippocampal long-term potentiation (LTP) and depression. Contextual fear memory was enhanced in the rats 1 week after SPS exposure, whereas LTP in the amygdala was blunted. Interestingly, blockade of GR activation by administering 17-beta-hydroxy-11-beta-/4-/[methyl]-[1-methylethyl]aminophenyl/-17-alpha-[prop-1-ynyl]estra-4-9-diene-3-one (RU40555), a GR antagonist, prior to SPS exposure prevented potentiation of fear conditioning and impairment of LTP in the CA1 region. Altogether, SPS caused a number of behavioral changes similar to those described in PTSD, which marks SPS as a putative PTSD model. The preventive effects of a GR antagonist suggested that GR activation might play a critical role in producing the altered behavior and neuronal function of SPS rats.

Intracellular channels.

Intracellular channels are located on the membranes of intracellular organelles and are involved in ion transfer, within the cytosolic compartments, in response to internal stimuli. Recently, various types of inositol 1,4,5-trisphosphate- and ryanodine-sensitive Ca(2+)-release channels, mitochondrial voltage-dependent anion channels, and a vesicular Cl- channel have been molecularly cloned and characterized, and their functional roles in the central nervous system are beginning to be clarified.

Involvement of transforming growth factor-beta and thrombopoietin in the pathogenesis of myelodysplastic syndrome with myelofibrosis.

We investigated the cause of myelofibrosis and proliferation of megakaryocytes in myelodysplastic syndrome with myelofibrosis (MDS-MF (+)). Plasma-transforming growth factor-beta1 (PTGF-beta1) concentrations closely correlated with myelofibrosis grade in MDS-MF (+) and were higher than those in idiopathic myelofibrosis (IMF), essential thrombocythemia (ET), idiopathic thrombocytopenic purpura (ITP), MDS-without MF (MDS-MF (-)) or healthy volunteers (HV). Peripheral blood mononuclear cells from MDS-MF (+) patients expressed more TGF-beta1 mRNA than those from IMF, MDS-MF (-) or HV. When we immunostained bone marrow specimens of MDS-MF (+) for TGF-beta, the intensity of blasts was apparently higher than that of megakaryocytes, while in MDS-MF (-), megakaryocytes were immunostained with a similar intensity as that in MDS-MF (+), but blasts were negative for staining. In IMF, megakaryocytes, monocytes and small mononuclear cells representing CD34+ cells were all similarly stained with a much lower intensity than that of blasts in MDS-MF (+). The number of bone marrow megakaryocytes were increased the most in MDS-MF (+), followed by ET, ITP, MDS-MF (-) and NHL and correlated with plasma thrombopoietin (TPO) levels or with plasma TGF-beta1 levels, respectively, in each disease. Thus, in MDS-MF (+), both myelofibrosis and the increased megakaryocytes were ascribed to overproduction of TGF-beta1 from blasts.

Induction of covalent DNA modifications and micronucleated erythrocytes by 4-nitroquinoline 1-oxide in adult and fetal mice.

Pregnancy and development are known to modify carcinogenesis. Little is known about the mechanism for the modulation. These studies investigated the relative sensitivity of nonpregnant, pregnant, and fetal mice to the induction of covalent DNA modifications and micronucleated erythrocytes by 4-nitroquinoline 1-oxide (4-NQO). Our results revealed that 4-NQO was bound to guanine nucleotides of DNA in all maternal and fetal organs tested. The adduct levels ranged from 2-60 base modifications per 10(9) DNA bases when 4-NQO was administered s.c. Overall, 4-NQO bound preferentially to DNA of the maternal tissues compared with that of the corresponding fetal tissues, with the exception of the liver. The adduct levels in maternal and fetal organs fell into 3 distinct levels. The greatest binding was in maternal lungs and pancreas (the target organs for carcinogenesis). The lowest binding levels were in maternal liver and all fetal organs studied. Gestation age at the time of 4-NQO treatment did not produce a significant effect on the amounts of adduct formation in the tissues examined, with the exception of placenta and bone marrow. Chronic treatment did not affect binding preference. At the cellular level, 4-NQO treatment induced twice the frequency of micronucleated erythrocytes in the bone marrow of pregnant mice compared with the nonpregnant mice and fetal liver, on a mg/kg basis. However, the polychromatic erythrocytes of fetal liver were more sensitive than those of adult bone marrow to the induction of micronuclei, when adduct levels were taken into account. A positive correlation of organotropsim between 4-NQO-induced DNA adducts and carcinogenicity was observed for maternal tissues, but not for fetal tissues. Fetal tissues, overall, lack the enzymes to metabolically activate 4-NQO. Fetal cells elicit greater biological responses, compared with adult cells, at equal adduct levels. This study reveals that the effective doses in maternal and fetal tissues may differ and, therefore, will be a better basis for further understanding the molecular mechanism of transplacental carcinogenesis.

Studies of high thermostability and peroxidase activity of recombinant antibody L chain-porphyrin Fe(III) complex.

A complex of an independent L chain from anti-mesotetrakis (4-carboxyphenyl) porphyrin (TCPP) monoclonal antibody 13-1 and TCPP Fe(III) was designated as L-zyme and shown to exhibit high peroxidase activity and high optimal reaction temperature (90 degrees C). Heat denaturation study and circular dichroism (CD) spectra analysis suggested that refolded structure of 13-1 L chain exhibited significantly reduced inactivation rate after heat treatment. The secondary structure of 13-1 L chain changed slightly by the encapsulation of TCPP Fe(III) and the complex was found to be less thermostable than the L chain alone. Furthermore, by characterization of truncated forms of the L chain, it was revealed that the hydrophobic region (115-146) and hydrophilic region (147-189) in CL are important for thermostability and activity, respectively. Tertiary structure of L-zyme was predicted by AbM. Comparison of residues of L-zyme with those in the active centre of known structure of the peroxidase from Arthromyces ramosus (ARP) indicated that His38(CDR1), His94(CDR3), Arg96(CDR3) of L-zyme are important residues for peroxidase activity. Moreover, the steric arrangements of these residues in both L-zyme and ARP are similar, respectively. Distance between proximal His and distal His in L-zyme is 9.09 A, whereas that of ARP is 7.8 A.

Thermostable peroxidase activity with a recombinant antibody L chain-porphyrin Fe(III) complex.

In order to engineer a new type of catalytic antibody, we attempt to use a monoclonal antibody L chain as a host protein for a porphyrin. TCPP (meso-tetrakis(4-carboxyphenyl)porphyine) was chemically synthesized and Balb/c mice were immunized using TCPP as a hapten. Two hybridoma cells (03-1, 13-1), that produce monoclonal antibody against TCPP, were obtained. Genes for both H and L chains of monoclonal antibodies were cloned, sequenced and overexpressed using E. coli as a host. ELISA and fluorescence quenching method show that the independent antibody L chains from both Mab03-1 and Mab13-1 have specific interaction with TCPP. Furthermore, the recombinant antibody L chain from Mab13-1 exhibits much higher peroxidase activity than TCPP Fe(III) alone. The enzyme activity was detectable with pyrogallol and ABTS (2,2-azinobis-3-ethylbenzthiazolin-6-sulfonic acid) but not with catechol. This new catalytic antibody was extremely thermostable. Optimum temperature of the peroxidase reaction by the complex of 13-1L chain and TCPP Fe(III) was 90 degrees C, while that the TCPP Fe(III) alone was 60 degrees C.

Inhibition of human O6-alkylguanine-DNA alkyltransferase and potentiation of the cytotoxicity of chloroethylnitrosourea by 4(6)-(benzyloxy)-2,6(4)-diamino-5-(nitro or nitroso)pyrimidine derivatives and analogues.

A series of 4(6)-(benzyloxy)-2,6(4)-diamino-5-(nitro or nitroso)pyrimidine derivatives and analogues of which 4(6)-benzyloxy groups were replaced with a (2-, 3-, or 4-fluorobenzyl)oxy or (2-, 3-, or 4-pyridylmethyl)oxy group, was synthesized. The abilities of these compounds to inhibit human O6-alkylguanine-DNA alkyltransferase (AGAT) in vitro and to potentiate the cytotoxicity of 1-[(4-amino-2-methyl-5-pyrimidinyl)methyl]-3-(2-chloroethyl) -3-nitrosourea (ACNU) toward HeLa S3 cells were evaluated. 2,4-Diamino-6-[(2-fluorobenzyl)oxy]-5-nitropyrimidine (3) and 2,4-diamino-5-nitro-6-(2-pyridylmethoxy)pyrimidine (6), whose ortho positions of the 6-substituent are modified, were much weaker in terms of these abilities than the corresponding meta- or para-modified compounds. These results are consistent with those of our previous study using a series of O6-benzylguanine derivatives. All 5-nitrosopyrimidine derivatives examined exerted both stronger AGAT-inhibition and ACNU-enhancement abilities than the corresponding 5-nitro derivatives. Among a variety of compounds that we have examined to date, 2,4-diamino-6-[(4-fluorobenzyl)oxy]-5-nitrosopyrimidine (10) exhibited the strongest ability to inhibit AGAT, and its magnitude was 2.5 and 50 times those of 4-(benzyloxy)-2,6-diamino-5-nitrosopyrimidine (9) and O6-benzylguanine (1), respectively. A strong positive correlation was observed between the ability to inhibit AGAT and to potentiate the cytotoxicity of ACNU. This strongly indicates that 4(6)-(benzyloxy)pyrimidine derivatives and their analogues potentiate ACNU cytotoxicity by inhibiting AGAT activity. To characterize the reactivity of test compounds, alkyl-transfer reactions were also carried out using the biomimetic alkyl-transfer system.

Long-term potentiation and long-term depression in hippocampal CA1 neurons of mice lacking the IP(3) type 1 receptor.

To investigate the role in synaptic plasticity of Ca(2+) released from intracellular Ca(2+) stores, mice lacking the inositol 1,4,5-trisphosphate type 1 receptor were developed and the physiological properties, long-term potentiation, and long-term depression of their hippocampal CA1 neurons were examined. There were no significant differences in basic synaptic functions, such as membrane properties and the input/output relationship, between homozygote mutant and wild-type mice. Enhanced paired-pulse facilitation at interpulse intervals of less than 60 ms and enhanced post-tetanic potentiation were observed in the mutant mice, suggesting that the presynaptic mechanism was altered by the absence of the inositol 1,4,5-trisphosphate type 1 receptor. Long-term potentiation in the field-excitatory postsynaptic potentials induced by tetanus (100 Hz, 1 s) and the excitatory postsynaptic currents induced by paired stimulation in hippocampal CA1 pyramidal neurons under whole-cell clamp conditions were significantly greater in mutant mice than in wild-type mice. Homosynaptic long-term depression of CA1 synaptic responses induced by low-frequency stimulation (1 Hz, 500 pulses) was not significantly different, but heterosynaptic depression of the non-associated pathway induced by tetanus was blocked in the mutant mice. Both long-term potentiation and long-term depression in mutant mice were completely dependent on N-methyl-D-aspartate receptor activity. To rule out the possibility of an effect compensating for the lack of the inositol 1,4,5-trisphosphate type 1 receptor occurring during development, an anti-inositol 1,4,5-trisphosphate type 1 receptor monoclonal antibody that blocks receptor function was diffused into the wild-type cell through a patch pipette, and the effect of acute block of inositol 1,4,5-trisphosphate type 1 receptor on long-term potentiation was examined. Significant enhancement of long-term potentiation was observed compared with after control immunoglobulin G injection, suggesting that developmental redundancy was not responsible for the increase in long-term potentiation amplitude observed in the mutant mouse. The properties of channels that could be involved in long-term potentiation induction were examined using whole-cell recording. N-methyl-D-aspartate currents were significantly larger in mutant mice than in wild-type mice only between holding potentials of -60 and -80 mV. We conclude that inositol 1,4,5-trisphosphate type 1 receptor activity is not essential for the induction of synaptic plasticity in hippocampal CA1 neurons, but appears to negatively regulate long-term potentiation induction by mild modulation of channel activities.

Ornithine decarboxylase activity and DNA synthesis in rats after long term treatment with butylated hydroxyanisole, sodium saccharin or phenobarbital.

Ornithine decarboxylase (ODC) activity and DNA synthesis were measured in the forestomach, urinary bladder and liver of rats given diet containing 2% butylated hydroxyanisole (BHA), 5% sodium saccharin (SS) or 0.05% phenobarbital (PB) for 21 weeks. ODC activity was not increased significantly in any of these organs in any of the groups, but the labeling index (LI) of the forestomach epithelium was increased by BHA and PB, and that of the urinary bladder epithelium by SS. PB did not increase the LI of the liver. These results do not indicate any relation between the effects of promoters, and the ODC activity or DNA synthesis in non-initiated target organs.

Wide difference between the cytotoxicity of the 11-alpha-and 11-beta-cyano analogues of tilivalline and their epimeric conversion.

Tilivalline (TV) possesses a pyrrolo[1,4]benzodiazepine nucleus and is cytotoxic toward mammalian cells. The 11-beta-cyano TV analogue (1) is about one hundred times more cytotoxic to mouse leukemia L1210 cells than TV itself. In contrast, the 11-alpha-cyano TV analogue (2), an epimer of 1, has only about one-hundredth the cytotoxicity of 1. It was found that epimerization proceeded between 1 and 2 under physiological conditions, and the cytotoxicity of 2 is thought to be caused mainly by 1 that was formed from 2 during incubation in medium.

Ulcerative colitis after autologous peripheral blood stem cell transplantation for non-Hodgkin's lymphoma.

A 54-year-old woman with peripheral T cell lymphoma in second complete remission (CR) received an autologous peripheral blood stem cell transplant (PBSCT). Antibiotic-resistant bloody diarrhea, and fever developed 110 days after transplant. Blood and stool cultures were negative. Skin rash was not observed. Barium enema and colonoscopy showed typical features of pancolonic-type ulcerative colitis (UC). Endoscopic biopsies confirmed the diagnosis of UC. Mesalazine and immunosuppressive therapy improved symptoms dramatically. We detected serum antibodies against synthetic tropomyosin (TM) peptide when UC was diagnosed. We postulate that autoimmunity including autoreactive anti-TM antibodies may be involved in the pathogenesis of UC after autologous PBSCT in this patient.

Long-term survival and differentiation of human peripheral blood CD34+ cells in SCID mice.

Human peripheral blood progenitor cells (PBPC) are currently used as a source of hematopoietic reconstitution by autologous transplantation after myeloabrative chemotherapy for malignancies. PBPC would also be useful for allogeneic transplantation since the collection of PBPC is much safer than that of bone marrow stem cells (BM). For allogeneic transplantation, it is imperative to confirm that PBPC contains self-renewable stem cells that can sustain a long lasting hematopoiesis. In the present study, we examined the reconstitution of human hematopoiesis in severe combined immunodeficiency (SCID) mice by transplanting peripheral blood CD34+ cells in which the neo gene was transduced as a marker. In 2 of 4 mice receiving PB-CD34+ cell transplantation, the neo gene appeared as early as 4 weeks and lasted as long as 24 weeks in all DNA preparations of bone marrow, peripheral blood and spleen cells from the SCID mice, while in 2 of 4 mice receiving BM-CD34+ cell transplantation, although the neo gene also lasted as late as 24 weeks, it did not appear as early as in the mice receiving PB-CD34+ cell transplantation. A similar observation was noted in clinical trials, i.e. the white blood cell and platelet recovered earlier by transplantation of PBPC than of BM. In mice who had the neo gene, we were also able to demonstrate by FACS the presence of human lineage specific antigen in the cells as late as 24 weeks after transplantation with PB-CD34+ cells, and the presence of human IgG in the sera 10 weeks after transplantation. These findings indicate that PB-CD34+ cells contain long-term repopulating stem cells which undergo differentiation in SCID mice.

Pulmonary veno-occlusive disease accompanied by microangiopathic hemolytic anemia 1 year after a second bone marrow transplantation for acute lymphoblastic leukemia.

Although hepatic veno-occlusive disease (HVOD) is a common complication of allogenic bone marrow transplantation (BMT), pulmonary veno-occlusive disease (PVOD) is very rare. Only three cases have been described in the literature. We report the case of a 19-year-old woman who developed PVOD accompanied by microangiopathic hemolytic anemia (MAHA) and hemolytic uremic syndrome (HUS) 1 year after a second BMT for relapsed acute lymphoblastic leukemia (ALL). Autopsy examination revealed obstruction of the small pulmonary veins with edematous thickening of the intima. These findings are compatible with PVOD. Pulmonary GVHD and pulmonary aspergillosis were also observed. Various etiologic factors have been implicated in PVOD after BMT. We postulate that pulmonary GVHD and pulmonary infection including aspergillosis played an important role in the occurrence of both PVOD and HUS in our patient. Microangiopathic cytokines released in response to the GVHD and infection may damage the intima of microvessels that were previously injured by the two BMT. Despite appropriate therapy, the microangiopathic process was irreversible and the patient died. Thus, measures must be taken to prevent and treat PVOD after BMT.

Use of PCR serum in diagnosing and monitoring cytomegalovirus reactivation in bone marrow transplant recipients.

We previously reported that the use of polymerase chain reaction (PCR) in detecting cytomegalovirus (CMV) DNA in serum (sPCR) enables the detection of CMV viremia, which has not been possible with other methods. In this study, the clinical usefulness of sPCR was investigated by comparison with the results of three other diagnostic methods, i.e., antigenemia assay (AG), shell vial culture test (shell vial), and complement-fixing (CF) antibody titer. The present study included 26 patients with hematological diseases who had undergone allogeneic bone marrow transplantation (BMT). A total of 347 samples were collected, and the results of the sPCR and AG methods were in agreement in 91.1% of the samples. When a subject was positive in both the sPCR and AG tests, and the other two tests (shell vial and CF) were also positive, CMV reactivation was surmised as definite. When only the result of the shell vial test or the CF test was positive, these results were taken as false-positives. The time at which the samples became positive in each of these four tests was 7.5 weeks post-BMT for sPCR, 7.0 weeks post-BMT for the AG test, 7.4 weeks post-BMT for the shell vial test, and 9.7 weeks post-BMT for the CF test. Thus, it was found that samples became positive at almost the same time for the sPCR, AG, and shell vial tests. Interstitial pneumonitis (IP) due to CMV developed in 3 subjects. These cases were positive in the sPCR, AG, and shell vial tests prior to the manifestation of symptoms of IP. The CF test did not become positive until after the onset of the disease. As the IP due to CMV was controlled with treatment, the sPCR and AG tests became negative. With the shell vial and CF tests, on the other hand, the test results continued to be positive even after the IP was cured. These findings demonstrate that the sPCR test method--like the AG test--yields few false-positive results. Therefore, the sPCR method is useful in early diagnosis of reactivation of CMV and for evaluation of the efficacy of therapy administered for IP. In addition, sPCR can be performed simultaneously on a large number of samples, and the evaluation of the test results is simple. We conclude that the sPCR test may be superior to the three other diagnostic methods for evaluation of serum samples from multiple institutions.

Long-term survival and late-onset complications of cancer patients treated with high-dose chemotherapy followed by autologous peripheral blood stem cell transplantation.

The antitumor effect of high-dose chemotherapy (HDC) followed by autologous peripheral blood stem cell transplantation (auto-PBSCT) is considered superior to that of conventional chemotherapy. However, the long-term benefits of this strategy in Japan remain unclear. Therefore, in this study, 109 cancer patients enrolled between 1989 and 1999 were treated with HDC and auto-PBSCT. Patients were evaluated for long-term survival and late-onset complications, including secondary malignancy. The mean number of CD34+ cells harvested per apheresis was larger in the group receiving high-dose cytosine arabinoside or high-dose etoposide plus granulocyte colony-stimulating factor (G-CSF) than in the group receiving conventional chemotherapy plus G-CSF. The 5-year overall survival rates for non-Hodgkin's lymphoma patients in first complete remission (CR) (83.2%), second or subsequent CR (74.1%), or first partial remission (PR) (66.7%) at the time of transplantation were significantly higher than those with no remission (35.7%) at the time of transplantation (first CR, P < .05; second or subsequent CR, P < .05; first PR, P < .05). The 5-year overall survival (OS) rates for breast cancer was 40.8%, and the disease-free survival rate was extremely low (8.8%). The 5-year OS rates for chemotherapy-sensitive and chemotherapy-resistant diseases at the time of transplantation were 32.7% and 35.7%, respectively, a difference that was not considered significant. The 5-year OS for germ cell tumor was 80.0%, and the disease-free survival rate was 77.9%. The rate of therapy-related death was 8.2%. The occurrence rate of secondary malignancy was 0.9%. Late-onset complications were observed in 4 cases (glomerulonephritis, interstitial pneumonitis, ulcerative colitis, and acute myelogenous leukemia). At 3.7%, the occurrence rate was not very high, but most complications of auto-PBSCT were life threatening and interfered with patients' quality of life. A careful follow-up is required for at least 2 years after transplantation, because the mean occurrence time of late-onset complications is 16.7 months posttransplantation.

Automated enzymatic determination of serum and urine creatine using the Abbott ABA-200.

A new enzymatic method is described for the determination of creatine in serum and urine with Abbott ABA-200. The measurement is accomplished by transforming creatine to formic acid in a reaction catalyzed by creatinase (creatine amidinohydrolase), sarcosine oxidase and formaldehyde dehydrogenase (see Figure 1). The assay takes less than 20 minutes. The standard response is linear for creatine concentrations up to 10 mg/dL (serum) and 80 mg/dL (urine). The coefficients of variation at 0.69 mg/dL (serum) and 4.93 mg/dL (urine) for within-day determination were less than 4% and, for between-day determination, were less than 5%. Results obtained by this procedure on one hundred serum and urine samples conformed well with a manual enzymatic method and the Folin method. The method is useful for the automated measurement of creatine in serum and urine.

A novel enzyme enantio-selectively synthesizes (R)salsolinol, a precursor of a dopaminergic neurotoxin, N-methyl(R)salsolinol.

In the human brain, only (R)enantiomer of 1-methyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline ((R)salsolinol) and N-methyl-salsolinol, a dopaminergic neurotoxin, were detected, suggesting their enzymatic biosynthesis. This paper reports the isolation and characterization of a novel enzyme, which enantio-selectively synthesizes (R)salsolinol from dopamine and acetaldehyde. Dopamine, acetaldehyde, formaldehyde and pyruvic acid were the substrates of this synthase, whereas N-methyldopamine, adrenaline, noradrenaline and L-DOPA were not. The possible function of this enzyme under physiological and pathological conditions in the brain is discussed.

A new enzymatic method to determine creatine.

A new enzymatic method is described for the determination of creatine in serum and urine using creatine amidinohydrolase (EC 3.5.3.3), sarcosine oxidase (EC 1.5.99.1) and formaldehyde dehydrogenase (EC 1.2.1.1). The principle of the method is as follows. Creatine is degraded to sarcosine and urea, and the sarcosine formed is measured with sarcosine oxidase in the presence of formaldehyde dehydrogenase and NAD+. The NADH + H+ produced is measured at 340 nm. Creatine concentration can be calculated directly from the absorptivity of NADH + H+ generated in the reaction or from creatine standard solutions. The assay takes less than 20 min. The standard curve is linear up to 50 mg creatine/1 (serum) and 800 mg creatine/1 (urine). Fifty random samples were assayed by this method (y) and simultaneously by the Folin method (x). The correlation coefficients were 0.995 for serum samples, 0.994 for urine samples, and the regression equations were y = 0.979x - 0.01 (serum) and y = 0.978x - 0.01 (urine).

Enhancement effect of O6-fluorobenzylguanines on chloroethylnitrosourea cytotoxicity in tumor cells.

O6-Alkylguanine derivatives sensitize tumor cells to chloroethylnitrosourea (CENU) chemotherapy by inactivation of O6-methylguanine-DNA methyltransferase (MGMT), which repairs CENU-induced O6-alkylguanines in DNA by accepting the alkyl group at a cysteine moiety. To test the biological significance of synthesized O6-fluorobenzylguanine derivatives, we measured their ability of inactivation of MGMT activity and their effects on the cytotoxicity of 1-(4-amino-2-methyl-5-pyrimidinyl) methyl-3-(2-chloroethyl)-3-nitrosourea hydrochloride (ACNU) in comparison with the effects of O6-benzylguanine and O6-phenylguanine. The O6-(4- and 3-fluorobenzyl)guanines considerably reduced the MGMT activity of HeLa S3 cell-free extract as did O6-benzylguanine. In contrast, O6-(2-fluorobenzyl)guanine and O6-phenylguanine had less of an effect on the activity. Two-hour pretreatment of O6-(4- and 3-fluorobenzyl) guanines potentiated ACNU cytotoxicity in HeLa S3 cells to a greater extent than did O6-(2-fluorobenzyl)guanine and O6-phenylguanine. The enhancement effects were consistent with the depletion of MGMT activity after the pretreatment of O6-fluorobenzylguanine derivatives. O6-Fluorobenzylguanines with a fluoro-substitution at the 4- or 3-position of the benzyl group were comparable to O6-benzylguanine and were powerful MGMT inactivators. The chemical features of the O6-benzyl group are a biologically important determinant in the reaction evolution with MGMT.