Protective effects of autophagy inhibitor 3-methyladenine on ischemia-reperfusion-induced retinal injury.

OBJECTIVE: To investigate the protective effects of autophagy inhibitor 3-methyladenine (3-MA) in a rat model of ischemic-reperfusion injury (IRI). METHODS: Forty Sprague-Dawley male rats (weight 220-250 g) were randomly divided into four groups: a control group (NC, n = 10), a Sham surgery group (n = 10), an IRI group (n = 10), and a 3-MA-treated IRI group [10 µL 3-MA (10 mmol/L) was injected in vitreous after the injury, n = 10]. The retinal IRI was induced by elevating the intraocular pressure to 110 mmHg for 60 min. Hematoxylin and eosin (HE) staining was used to calculate the number of retinal ganglion cells (RGCs). The level of microtubule-associated protein 1A/1B light chain 3 (LC3), Beclin-1, and Caspase-3 in the retina was detected using the immunofluorescence staining method. The LC3, Beclin-1, B-cell lymphoma/leukemia-2 (Bcl-2), and Caspase-3 protein levels were examined by Western blotting. RESULTS: The number of RGCs in IRI group was significantly lower than that in NC group (P < 0.05), demonstrated by HE staining. Western blotting results indicated that the protein expression of LC3 and Beclin-1 in the IRI group was significantly elevated compared with those in the NC group (P < 0.05). However, with 3-MA treatment, the number of RCGs in 3-MA-treated IRI group was elevated and protein levels of LC3, Beclin-1 were down-regulated, compared with those in the IRI group (P < 0.05). Further immunohistochemistry staining and Western blot showed that 3-MA-treated IRI group presented down-regulated Caspase-3 and up-regulated Bcl-2 protein expression with comparison of IRI group (P < 0.05). CONCLUSIONS: Retina IRI-caused RGCs loss involved activated autophagy pathway and apoptosis, which could be prevented by autophagy inhibitor 3-MA. Autophagy inhibitor 3-MA may act as a potent therapeutic tool in attenuating retina IRI.

Interleukin­17A facilitates osteoclast differentiation and bone resorption via activation of autophagy in mouse bone marrow macrophages.

Interleukin 17A (IL­17A) exerts pleiotropic effects on periodontitis, partially through enhancement of alveolar bone loss. Osteoclasts are the main culprits that absorb alveolar bone. However, studies describing the correlation between IL­17A and osteoclasts are not conclusive. Previously, autophagy was revealed to be involved in osteoclast differentiation and bone resorption. However, the role of autophagy in IL­17A­mediated osteoclast formation is yet to be clarified. In the present study, bone marrow macrophages (BMMs) were treated with or without IL­17A. 3­Methyladenine (3­MA) was applied to inhibit autophagy. Osteoclast formation was detected by tartrate­resistant acid phosphatase (TRAP) staining, immunofluorescence, and scanning electron microscope. The effects of IL­17A on osteoclast­specific genes and autophagy­related genes during osteoclast differentiation were examined by real­time quantitative polymerase chain reaction and western blot analysis. Autophagosomes were observed by transmission electron microscope. Hematoxylin and eosin (H&E), and TRAP staining was adopted to assess alveolar bone destruction and the number of osteoclasts, respectively in a rat periodontitis model. Consequently, IL­17A stimulated osteoclast differentiation and bone resorption of BMMs accompanied by an increase in the mRNA expression of osteoclast­specific genes. Furthermore, IL­17A increased the levels of autophagy­related genes and proteins, and inhibition of autophagy with 3­MA attenuated the IL­17A­mediated osteoclastogenesis. In addition, there was an increase in the number of osteoclasts and alveolar bone resorption with IL­17A treatment in the periodontitis rat model. Collectively, these findings indicated that IL­17A facilitated osteoclast differentiation and bone resorption in vitro and in vivo, which may contribute to the understanding of the molecular basis of IL­17A in alveolar bone destruction and provide insight on the clinical therapeutic targets for periodontitis.

Schisandrin C enhances odontoblastic differentiation through autophagy and mitochondrial biogenesis in human dental pulp cells.

OBJECTIVE: To investigate the role of Schisandrin C in odontoblastic differentiation, and its relations between autophagy and mitochondrial biogenesis in human dental pulp cells (HPDCs). DESIGN: Fresh third molars were used, and cultured for HDPCs. Western blotting technique, Alizarin red S staining, alkaline phosphatase (ALP) activity, and confocal microscopy were used to detect autophagy, mitochondrial biogenesis, and odontoblastic differentiation. To understand the mechanism of Schisandrin C, the HDPCs were treated with lipopolysaccharide (LPS), autophagy and heme oxygenase-1 (HO-1) inhibitors: 3-Methyladenine (3-MA) and Zinc protoporphyrin IX (ZnPP), respectively. RESULTS: LPS decreased the expression of autophagy molecules [autophagy protein 5 (ATG-5), beclin-1, and microtubule-associated protein 1A/1B light chain 3 (LC3-I/II)] and mitochondrial biogenesis molecules [heme oxygenase-1 (HO-1) and peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α)], and disrupted odontoblastic differentiation. The down-regulation of autophagy and mitochondrial biogenesis with 3-MA and ZnPP inhibited odontoblastic differentiation. However, Schisandrin C restored the expression of all the above molecules, even with LPS and inhibitor treatment. This result demonstrates that autophagy and mitochondrial biogenesis plays an essential role in odontoblastic differentiation, and Schisandrin C activates these systems to promote odontoblastic differentiation of HDPCs. CONCLUSION: Schisandrin C has potential characters to regulate odontoblastic differentiation, and may be recommended for use as a compound for pulp homeostasis.

Metabolic activation of pradefovir by CYP3A4 and its potential as an inhibitor or inducer.

Metabolic activation of pradefovir to 9-(2-phosphonylmethoxyethyl)adenine (PMEA) was evaluated by using cDNA-expressed CYP isozymes in portal vein-cannulated rats following oral administration and in human liver microsomes. The enzyme induction potential of pradefovir was evaluated in rats following multiple oral dosing and in primary cultures of human hepatocytes. The results indicated that CYP3A4 is the only cDNA-expressed CYP isozyme catalyzing the conversion of pradefovir to PMEA. Pradefovir was converted to PMEA in human liver microsomes with a K(m) of 60 microM, a maximum rate of metabolism of 228 pmol/min/mg protein, and an intrinsic clearance of about 359 ml/min. Addition of ketoconazole and monoclonal antibody 3A4 significantly inhibits the conversion of pradefovir to PMEA in human liver microsomes, suggesting the predominant role of CYP3A4 in the metabolic activation of pradefovir. Pradefovir at 0.2, 2, and 20 microM was neither a direct inhibitor nor a mechanism-based inhibitor of CYP3A4, CYP2D6, CYP2C9, CYP2C19, CYP2E1, and CYP1A2 in human liver microsomes. In rats, the liver was the site of metabolic activation of pradefovir, whereas the small intestine did not play a significant role in the metabolic conversion of pradefovir to PMEA. Daily oral dosing (300 mg/kg of body weight) to rats for 8 days showed that pradefovir was not an inducer of P450 enzymes in rats. Furthermore, pradefovir at 10 microg/ml was not an inducer of either CYP1A2 or CYP3A4/5 in primary cultures of human hepatocytes.

Magnesium, essential for base excision repair enzymes, inhibits substrate binding of N-methylpurine-DNA glycosylase.

N-Methylpurine-DNA glycosylase (MPG) initiates base excision repair in DNA by removing a wide variety of alkylated, deaminated, and lipid peroxidation-induced purine adducts. MPG activity and other DNA glycosylases do not have an absolute requirement for a cofactor. In contrast, all downstream activities of major base excision repair proteins, such as apurinic/apyrimidinic endonuclease, DNA polymerase beta, and ligases, require Mg(2+). Here we have demonstrated that Mg(2+) can be significantly inhibitory toward MPG activity depending on its concentration but independent of substrate type. The pre-steady-state kinetics suggests that Mg(2+) at high but physiologic concentrations decreases the amount of active enzyme concentrations. Steady-state inhibition kinetics showed that Mg(2+) affected K(m), but not V(max), and the inhibition could be reversed by EDTA but not by DNA. At low concentration, Mg(2+) stimulated the enzyme activity only with hypoxanthine but not ethenoadenine. Real-time binding experiments using surface plasmon resonance spectroscopy showed that the pronounced inhibition of activity was due to inhibition in substrate binding. Nonetheless, the glycosidic bond cleavage step was not affected. These results altogether suggest that Mg(2+) inhibits MPG activity by abrogating substrate binding. Because Mg(2+) is an absolute requirement for the downstream activities of the major base excision repair enzymes, it may act as a regulator for the base excision repair pathway for efficient and balanced repair of damaged bases, which are often less toxic and/or mutagenic than their subsequent repair product intermediates.

Inhibition of mRNA deadenylation and degradation by ultraviolet light.

Post-transcriptional mechanisms contribute to the changes in gene expression induced by cell stress. The effect of UV-B light on mRNA degradation in HeLa cells was investigated using a transcriptional chase system to determine the decay kinetics of tet-off vector-derived mRNAs containing or lacking a destabilizing AU-rich element. Degradation of both mRNAs was strongly inhibited in cells exposed to UV-B light. Removal of the poly(A)-tail, considered a crucial step in mRNA degradation, was strikingly impaired. UV light also inhibited deadenylation and degradation of endogenous mRNA of the chemoattractant cytokine interleukin (IL)-8. Both effects occurred rapidly and independently of newly induced genes. Importantly, stabilization of IL-8 mRNA was accompanied by a strong increase in the duration of IL-8 protein formation. Furthermore, general inhibition of protein synthesis, a hallmark of the response to cell stress, required far higher doses of UV-B than inhibition of mRNA deadenylation and degradation. The difference in sensitivity of cells to these effects of UV-B light establishes a dose range in which mRNA stabilization can lead to dramatically enhanced expression of proteins derived from normally unstable mRNAs, such as those of inflammatory cytokines, growth factors and proto-oncogenes, and thereby have a major impact on the response to UV light.

Adenosine and deoxyadenosine induces apoptosis in oestrogen receptor-positive and -negative human breast cancer cells via the intrinsic pathway.

In this study we have examined the cytotoxic effects of different concentrations of adenosine (Ado) and deoxyadenosine (dAdo) on human breast cancer cell lines. Ado and dAdo alone had little effect on cell cytotoxicity. However, in the presence of adenosine deaminase (ADA) inhibitor, EHNA, adenosine and deoxyadenosine led to significant growth inhibition of cells of the lines tested. Ado/EHNA and dAdo/EHNA-induced cell death was significantly inhibited by NBTI, an inhibitor of nucleoside transport, and 5'-amino-5'-deoxyadenosine, an inhibitor of adenosine kinase, but the effects were not affected by 8-phenyltheophylline, a broad inhibitor of adenosine receptors. The Ado/EHNA combination brought about morphological changes consistent with apoptosis. Caspase-9 activation was observed in MCF-7 and MDA-MB468 human breast cancer cell lines on treatment with Ado/EHNA or dAdo/EHNA, but, as expected, caspase-3 activation was only observed in MDA-MB468 cells. The results of the study, thus, suggest that extracellular adenosine and deoxyadenosine induce apoptosis in both oestrogen receptor-positive (MCF-7) and also oestrogen receptor-negative (MDA-MB468) human breast cancer cells by its uptake into the cells and conversion to AMP (dAMP) followed by activation of nucleoside kinase, and finally by the activation of the mitochondrial/intrinsic apoptotic pathway.

Lack of antagonism between abacavir, lamivudine, and tenofovir against wild-type and drug-resistant HIV-1.

Potential contributors to the high rate of virologic failure observed for tenofovir, abacavir, and lamivudine include a low genetic barrier to resistance for this regimen and antagonistic drug-drug interactions. To examine the second possibility, we tested combinations of abacavir, tenofovir, and lamivudine against wild-type and drug-resistant HIV-1 in vitro using peripheral blood mononuclear cells and MT-4 cells. Antagonistic interactions were not detected for any combination. If the systems examined accurately reflect the in vivo situation, antagonism does not substantially contribute to the poor efficacy of this triple combination.

Hydrolysis of N-methyl-D-aspartate receptor-stimulated cAMP and cGMP by PDE4 and PDE2 phosphodiesterases in primary neuronal cultures of rat cerebral cortex and hippocampus.

Stimulation of N-methyl-D-aspartate (NMDA) receptors on neurons activates both cAMP and cGMP signaling pathways. Experiments were carried out to determine which phosphodiesterase (PDE) families are involved in the hydrolysis of the cyclic nucleotides formed via this mechanism, using primary neuronal cultures prepared from rat cerebral cortex and hippocampus. The nonselective PDE inhibitor 3-isobutyl-1-methylxanthine (IBMX) potentiated the ability of NMDA to increase cAMP and cGMP. However, among the family-selective inhibitors, only the PDE4 inhibitor rolipram enhanced the ability of NMDA to increase cAMP in the neurons. In contrast, only the PDE2 inhibitor erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA) enhanced the ability of NMDA to increase cGMP. Neither adenosine nor an adenosine deaminase inhibitor mimicked the effect of EHNA; this suggests that EHNA's inhibition of PDE2, not its effects on adenosine metabolism, mediates its effects on NMDA-stimulated cGMP concentrations. The PDE inhibitor-augmented effects of NMDA on cAMP and cGMP formation were antagonized by 5-methyl-10,11-dihydro-5H-dibenzo[a,d] cyclohepten-5,10-imine maleate (MK-801), verifying NMDA receptor mediation. In contrast, only NMDA-mediated cGMP formation was affected by altering either nitric oxide signaling or guanylyl cyclase; this suggests that NMDA-induced changes in cAMP are not secondary to altered cGMP concentrations. Overall, the present findings indicate that cAMP and cGMP formed in neurons as a result of NMDA receptor stimulation are hydrolyzed by PDE4 and PDE2, respectively. Selective inhibitors of the two PDE families will differentially affect the functional consequences of activation of these two signaling pathways by NMDA receptor stimulation.

Cytotoxicity of antiviral nucleotides adefovir and cidofovir is induced by the expression of human renal organic anion transporter 1.

The transport of organic anions in proximal convoluted tubules plays an essential role in the active secretion of a variety of small molecules by the kidney. In addition to other anionic substrates, the human renal organic anion transporter 1 (hOATI) is capable of transporting the nucleotide analogs adefovir and cidofovir. To investigate the involvement of hOATI in the mechanism of nephrotoxicity associated with these two clinically important antiviral agents, Chinese hamster ovary (CHO) cells were stably transfected with hOATI cDNA. The resulting CHOhOAT cells showed probenecid-sensitive and pH-dependent uptake of p-aminohippurate (Km = 15.4 FtM, V,,, ..ax = 20.6 pmol/106 cells min), a prototypical organic anion substrate. In addition, the stably expressed hOATI mediated efficient transport of adefovir (Km, = 23.8 tLM, V, a,, = 46.0 pmol/106 cells min) and cidofovir (K, = 58.0 /iM, Vt,ax = 103 pmol/106 cells * min) such that the levels of intracellular metabolites of both nucleotides were > 1 00-fold higher in CHOh OAT cells than in parental CHO. Consequently, adefovir and cidofovir were approximately 500-fold and 400-fold more cytotoxic, respectively, in CHOh OAT cells compared to CHO. The cytotoxicity of both drugs in CHOh OAT cells was markedly reduced in the presence of hOATI inhibitors. The cyclic prodrug of cidofovir, which exhibits reduced in vivo nephrotoxicity, was a poor substrate for hOATI and showed only marginally increased cytotoxicity in CHOh OAT cells. In conclusion, these studies demonstrate that hOATI plays a critical role in the organ-specific toxicity of adefovir and cidofovir, and indicates that CHOh OAT cells may represent a useful in vitro model to investigate the potential nephrotoxicity of clinically relevant organic anion agents.

Inhibition of 6-methyladenine formation decreases the translation efficiency of dihydrofolate reductase transcripts.

Cycloleucine was used to inhibit the formation of internal N6-methyladenosine residues in the messenger ribonucleic acid transcripts from cultured methotrexate resistant mouse sarcoma cells. Cells cultured in cycloleucine produced transcripts deficient in N6-methyladenosine residues and the 2'-O-methylated nucleosides of the cap structure; however, the formation of the 7-methylguanine nucleoside of the cap was not effected. Cytoplasmic polyadenylated transcripts were isolated from cells which had been pretreated with media containing cycloleucine and translated in an in vitro translation assay. The levels of translated dihydrofolate reductase were then analyzed by polyacrylamide gel electrophoresis. The amount of dihydrofolate reductase protein produced from the transcripts of the cycloleucine treated cells was 20% less than untreated transcripts. Ribonuclease protection assays demonstrated little difference in the cytoplasmic levels of dihydrofolate reductase transcripts between treated and untreated cells suggesting that the decrease in translation efficiency was not caused solely by an alteration in the processing or cytoplasmic transport of the transcripts. Translation of in vitro transcribed transcripts showed the presence of 2'-O-methylated nucleosides in the cap structure had a negative effect on translation efficiency, demonstrating that the results observed from cycloleucine treatment could not be due to the inhibition of 2'-O-methylation in the cap. These experiments therefore suggest that an inhibition of N6-methyladenosine residues in dihydrofolate reductase transcripts significantly alters their rate of translation.

Inhibition of intestinal metabolism of the antiviral ester prodrug bis(POC)-PMPA by nature-identical fruit extracts as a strategy to enhance its oral absorption: an in vitro study.

PURPOSE: To explore the usefulness of fruit extracts as enhancers of the oral absorption of esterase-sensitive prodrugs. METHODS: Inhibition of esterase-mediated degradation by nature-identical fruit extracts was evaluated using 1) p-nitrophenylacetate (model substrate for esterase-activity) in rat intestinal homogenates and 2) bis(isopropyloxycarbonyloxymethyl)-(R)-9-[(2-phosphonomethoxy++ +)propyl]adenine [bis(POC)-PMPA] (esterase-sensitive prodrug of the antiviral agent PMPA) in Caco-2 cell homogenates and in intestinal homogenates from rat, pig and man. Subsequently, transport of the ester prodrug was studied across Caco-2 monolayers in the presence or absence of fruit extracts. RESULTS: In homogenates from rat ileum, the esterase activity could be reduced significantly by the inclusion of fruit extracts (1%): the initial enzymatic degradation of p-nitrophenylacetate was inhibited by 77% (strawberry), 16% (passion fruit) and 57% (banana). A similar inhibition of bis(POC)-PMPA metabolism by fruit extracts was observed in intestinal homogenates from several species and in homogenates from Caco-2 cells. Transport of total PMPA across Caco-2 monolayers was enhanced 3-fold by co-incubation with strawberry extract (1%). The fraction of intact prodrug appearing in the acceptor compartment increased from virtually zero to 67%. CONCLUSIONS: The results suggest that co-incubation with nature-identical fruit extracts might be useful as a strategy to enhance the transepithelial transport of esterase-sensitive prodrugs through inhibition of intracellular metabolism of the prodrug.

Intravascular adenosine: the endothelial mediators of its negative dromotropic effects.

Intravascular adenosine may exert its negative dromotropic effect via activation of luminal coronary endothelial receptors, which suggests the presence of transcellular dromotropic mediators of endothelial origin, perhaps nitric oxide (NO) and prostaglandins. We decided to test this hypothesis in isolated guinea pig hearts retrogradely perfused with Krebs-Henseleit solution. A pair of stimulating electrodes were placed in the right atria and the auricular-ventricular (A-V) delay recorded by means of a recording electrode placed on the left atria and an electrode placed on the tip of the ventricle. Hearts were paced at a rate of 3.8 +/- 0.2 Hz and perfused at a coronary flow of 9 +/- 0.25 ml/min. To obtain dose-response curves, single doses (as boluses) of different concentrations of adenosine were infused and the maximal increase in A-V delay induced by each dose was determined. Agents that inhibit NO accumulation, such as N(G)-nitro-L-arginine methyl ester (L-NAME) and oxyhemoglobin, diminished the effect of adenosine while NO-sparing agents, such as superoxide dismutase and dithiotreitol, enhanced the adenosine effect. Infusion of NO and the NO donor morpholinosydnonimine increased the A-V delay in a dose-dependent manner. In addition, the dose-response curve for adenosine was displaced downward and to the right by indomethacin, indicating also the involvement of prostaglandins. Infusion of L-NAME in addition to indomethacin further diminished the effects of adenosine, indicating that NO and prostaglandins acted simultaneously. To selectively activate intravascular endothelial adenosine receptors, adenosine amino congener (ADAC), an adenosine A1 receptor agonist, was covalently coupled to 2 X 10(6) Da dextran. When intracoronarily infused, the dextran-ADAC complex remains in the blood vessel lumen because it is too large to diffuse to the interstitium. On intracoronary administration, the dextran-ADAC complex caused a negative dromotropic effect which was diminished by L-NAME and indomethacin. Our data indicate that the dromotropic effect caused by intracoronarily administered adenosine is the result solely of activation of intravascular endothelial adenosine receptors, possibly type A , and that NO and prostaglandins are synergistic endothelial mediators of this effect.

Effects of adenosine deaminase inhibition on blood pressure in old spontaneously hypertensive rats.

Adenosine is an ubiquitously occurring endogenous nucleoside that via cell surface receptors exerts multiple antihypertensive actions, and mediates a number of biological responses that may reduce cardiovascular disease risk. Therefore modulation of endogenous levels of adenosine may offer beneficial effects in hypertension. The objective of this study was to determine whether inhibition of adenosine deaminase lowers blood pressure in spontaneously hypertensive rats (SHR). We investigated the effects of erythro-9-(2-hydroxyl-3-nonyl) adenine (EHNA), an adenosine deaminase inhibitor, on hemodynamic and renal parameters in 16-week-old and 36-week-old SHR and normotensive Wistar Kyoto rats (WKY) and in 36-week-old SHR and WKY pretreated with 1,3-dipropyl-8-p-sulfopheznylxanthine (DPSPX, an adenosine antagonist that does not enter the brain and is restricted to the extracellular space). Adenosine deaminase inhibition with EHNA (10 mg/kg, iv.) produced a marked fall in arterial blood pressure in older (MABP 162.0+/-7.6 mmHg and 120.7+/-11.7 mmHg for baseline and EHNA period, respectively; p<0.01), but not younger, SHR, whereas no effects on blood pressure were observed in age-matched normotensive WKY rats. EHNA did not affect renal hemodynamic and excretory function in any of six groups of animals. DPSPX blocked the antihypertensive effects of EHNA, suggesting that the effects of EHNA on blood pressure are mediated via peripheral adenosine receptors. Further studies are required to elucidate why inhibition of adenosine deaminase lowers blood pressure only in older SHR. The present data suggest that inhibition of adenosine deaminase may provide beneficial effects in older hypertensives and lead us to propose that design and use of extracellular adenosine deaminase inhibitors may offer cardiovascular protection in hypertension.

Oocyte maturation in rabbits: effects of calmodulin inhibitors.

Oocyte maturation in mammals follows a highly conserved pattern of release from arrest through to the extrusion of the first polar body and formation of the second metaphase spindle. Oscillations in cytoplasmic calcium concentration precede the events of maturation in many species. These calcium ions interact with and activate calcium-binding proteins, including calmodulin, within the cell. Thus, it was of interest to us to examine whether calcium acted through calmodulin in the initial stages of maturation in rabbit oocytes or whether calmodulin was required for continuation through metaphase I no to metaphase II. Using the calmodulin inhibitor W-7 we found a significant (p < 0.05) decrease in the percentage of oocytes that underwent germinal vesicle breakdown. Calmidazolium did not prevent germinal vesicle breakdown; however, it caused a significant (p < 0.05) decrease in the proportion of oocytes with fully elaborated spindles and taxol-induced cytoplasmic asters. Both inhibitors caused a significant (p < 0.05) reduction in the proportion of oocytes that extruded their first polar bodies. The kinase inhibitor 6-DMAP caused a significant reduction in the proportion of oocytes with spindles and condensed chromatin, indicating the necessity for phosphorylation events in the resumption of meiosis. In rabbit oocytes calmodulin may play a role in the release from prophase arrest, and it is necessary for spindle preservation and continuation through metaphase I to metaphase II. The varying effects of the two inhibitor stems from their different binding sites on the calmodulin molecule thus causing a differential effect on its downstream effectors.

Purification and structural determination of an inhibitor of starfish oocyte maturation from a Bacillus species.

Inhibitors of bacterial origins of starfish oocyte maturation were sought to obtain biologically active substances which act on either hormonal signal transduction or cell cycle regulation. An oocyte maturation-inhibiting substance found in culture fluid of a Bacillus species was purified to homogeneity. This substance possessed the nature of a detergent and specifically inhibited 1-methyladenine-induced oocyte maturation (50% inhibitory concentration, 3.3 microM) but not dithiothreitol-induced maturation. Its total structure was established to be the lactone of 3-hydroxy-13-methyltetradecanoyl-Glu-Leu-Leu-Val-Asp-Leu -Leu through COOH of the carboxy-terminal Leu. This structure is identical to surfactin, although although the configuration of the substance's amino acid residues has not yet been determined. Surfactin was shown to be identical with this substance in its inhibitory effect on starfish oocyte maturation as well as its chromatographic and electrophoretic properties. Therefore, it was concluded that the oocyte maturation-inhibiting substance produced by a Bacillus species is surfactin.

Nephrotoxicity induced by a single dose of adenine: effects of 4-aminopyrazolo[3,4-d]pyrimidine and allopurinol.

The effects of allopurinol and 4-aminopyrazolo[3,4-d]pyrimidine (4APP) on adenine-induced renal injury in mice were examined. Plasma urea nitrogen (UN) and creatinine levels increased after the oral administration of adenine to mice. However, plasma UN and creatinine levels decreased inversely with the dose of 4APP when a different dosage of 4APP was administered together with adenine. Yet 4APP did not have any effect on the UN or creatinine levels when 4APP was administered after adenine administration. Plasma UN and creatinine levels increased in the allopurinol-administered group as in the adenine-administered group. Moreover, from light microscopic observation by hematoxylin-eosin staining, microvacuolic changes in the proximal tubuli were detected in the mouse kidney in the adenine-administered group, and epithelial cell loss, degeneration and microvacuolic changes in the proximal tubuli were observed in the mouse kidney in the adenine-and-allopurinol-administered group. However, there were no changes in the proximal tubuli in the mouse kidney in the adenine-and-4APP-administered group. These findings suggested that 4APP inhibits the action of adenine in the mouse kidney.

Okadaic acid accelerates germinal vesicle breakdown and overcomes cycloheximide- and 6-dimethylaminopurine block in cattle and pig oocytes.

Pig and cattle oocytes, when released from the follicle, spontaneously resume first meiotic division within 20 or 8 hr, respectively. In oocytes of both species, the activity of histone H1 kinase increases during maturation, exhibiting a maximum in metaphase I. Treatment of these oocytes with okadaic acid results in acceleration of germinal vesicle breakdown (GVBD) and of histone H1 kinase activation. This effect is more important in pig oocytes, in which the acceleration rises for 6 hr, as compared to 2 hr in cattle. Moreover, under these conditions, H1 kinase activity measured after 12 hr of culture appears higher than that observed in control metaphase I oocytes. When added to prophase oocytes, both cycloheximide and 6-DMAP (6-dimethylaminopurine) block GVBD and histone H1 kinase activation. Okadaic acid, at a concentration of 2.5 microM, is able to release the inhibitory effect exerted by cycloheximide on histone H1 kinase activity; however, GVBD occurred only in two-thirds of pig and one-quarter of cattle oocytes after 20 hr of culture. In addition, okadaic acid fully reverses the effect of 6-DMAP on H1 kinase activity and on GVBD in both species. The opposite effects of 6-DMAP and okadaic acid on MPF activation are discussed, as well as the nature of the protein, which has to be synthesized during the first meiotic division and may be involved in the MPF activation cascade.

Purine base transport in wild-type and mycophenolic acid-resistant Tritrichomonas foetus.

The purine base transport systems of wild-type and mycophenolic acid-resistant (MPAR) Tritrichomonas foetus have been characterized. Wild-type T. foetus has two carriers, one for hypoxanthine (Km = 0.7 +/- 0.3 mM, Vm = 80 +/- 20 pmol microliters-1min-1) and guanine (Km = 0.09 +/- 0.02 mM, Vm = 17 +/- 3 pmol microliters-1min-1), and a second for xanthine (Km = 0.6 +/- 0.2 mM, Vm = 25 +/- 5 pmol microliters-1min-1). Adenine transport was not saturable (k = 0.16 +/- 0.01 min-1) and therefore appears to enter the parasite by passive diffusion through the membrane. T. foetus MPAR has lost the hypoxanthine/guanine transporter. Xanthine and adenine transport are similar in wild-type and MPAR T. foetus. No purine nucleoside transporter could be identified.

Toxoplasma gondii: in vivo and in vitro studies of a mutant resistant to arprinocid-N-oxide.

The anticoccidial drug arprinocid and arprinocid-N-oxide, a metabolite produced in vivo, blocked the growth of Toxoplasma gondii in human fibroblasts. The more potent arprinocid-N-oxide inhibited growth by 50% at 20 ng/ml while arprinocid inhibited at 2 micrograms/ml. For both drugs, the host cell was less sensitive than was the parasite. Hypoxanthine did not reverse the antitoxoplasma activity of either drug. We isolated a parasite mutant, R-AnoR-1 that was 16- to 20-fold more resistant to arprinocid-N-oxide than was the wild type RH T. gondii. This mutant was not resistant to arprinocid in vitro. Arprinocid in a daily oral dose of 136 micrograms regularly protected mice against an otherwise fatal infection with RH T. gondii and 55 micrograms had some protective effect. However, all mice infected with R-AnoR-1 and treated with 360 micrograms arprinocid per day died. Since this mutant is fully sensitive to arprinocid, the form of the drug that is therapeutically active in vivo cannot be arprinocid and is likely to be arprinocid-N-oxide.