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Since the late 1970’s, extensive testing has been conducted at Gramercy to remove high molecular weight organic compounds (humates) from Bayer process liquors. Early research work tested the removal of humates using the Giulini process (Dolime) , and by the use of organic polymeric additives.

Zona pellucida hardening is a natural process that occurs after oocyte fertilization to prevent polyspermic fertilization and to protect embryonic development. Pre-fertilization hardening of the zona pellucida, however, decreases fertilization rates. Cryoprotectants have also been shown to negatively affect fertilization rates, one possible mechanism of which being through zona hardening. This experiment was conducted to determine the effect of different cryoprotectants on hardening of the zona pellucida of mature bovine oocytes. Oocytes were collected by ovum pick-up (OPU) by transvaginal ultrasound guided aspiration (TUGA) from mixed-breed cows. After collection, oocytes were randomly assigned to 3 cryoprotectant treatment groups: dimethyl sulfoxide (DMSO), glycerol, or PBS (control). Drops (50 µL) of each vitrification solution were placed under mineral oil. Vitrification solution 1 (VS1) contained 10% ethylene glycol (EG), either 10% DMSO or glycerol, and 0.5 M sucrose. Vitrification solution 2 (VS2) contained 20% EG, 20% DMSO or glycerol, and 0.5 M sucrose. All oocytes were held in VS1 for 5 min before being transferred to VS2 for 45 s. All oocytes were washed in a common dilution solution (80% PBS, 20% calf serum, 0.025 M sucrose) for 5 min. Next, oocytes were moved to 50-µL drops of protease solution (0.1% protease) under mineral oil. Control oocytes were held in PBS for ~10 min before entering the protease solution to represent the same period as the vitrification procedure. The oocytes were observed until the zonae pellucidae were completely digested and times were recorded for each oocyte. This experiment included 4 replicates with a total of 88 oocytes used, 32 each in DMSO and glycerol and 24 in PBS. The data were analysed using ANOVA. The DMSO group had the lower mean zona digestion time out of the 2 cryoprotectants at 15.75 min and glycerol had the highest mean digestion time at 19.3 min. The control group (PBS) had the lowest mean of the 3 treatments at 12.7 min. The differences between DMSO and glycerol, and between DMSO and PBS were not significant (P = 0.0654 and 0.1073, respectively). However, both glycerol versus PBS and the average of DMSO and glycerol versus PBS were significantly different (P-value = 0.0053 and 0.0119, respectively). These results suggest that glycerol hardens the zona pellucida more than DMSO or PBS; however, there is not enough evidence to determine whether DMSO hardens the zona pellucida compared with PBS. This would suggest that, in relation to zona hardening and ensuring proper fertilization, glycerol-based cryoprotectants may be a better option than DMSO-based ones. Further, these results may be important in embryo vitrification as zona hardening may prevent blastocyst hatching, suggesting that glycerol-based cryoprotectants should be investigated as the optimal cryoprotectant here also.

Fetal disorders of mitochondrial fatty acid oxidation have recently been associated with obstetric complications including pre-eclampsia, Hemolysis, Elevated Liver enzymes, Low Platelets (HELLP) syndrome, placental floor infarct, and Acute Fatty Liver of Pregnancy (AFLP). These diseases occur in about a third of the mothers who are heterozygous for a defect in long chain 3-hydroxyacyl-CoA dehydrogenase (LCHAD) enzyme and who bear a fetus homozygous for the defect. The mechanism of this association is not clearly understood. In this study, we provide evidence that the placenta may be the site of production of toxic intermediates of fatty acid metabolism, which accumulate to cause liver damage in the mother. We show that two critical enzymes of long chain fatty acid metabolism, long chain 3-hydroxyacyl-CoA dehydrogenase (LCHAD) and short chain 3-hydroxyacyl-CoA dehydrogenase (SCHAD), are active in the normal human placenta. There is an inverse correlation between the enzyme activity of both the enzymes and maternal gestational age during the second and third trimesters. We believe that the demonstration of fatty acid oxidation enzyme activity by the placenta is the first step towards assessing a possible role for fetal/placental fatty acid oxidation defects in the pathogenesis of a subset of pregnancy complications.

While vitrification has become a valuable system used in oocyte and embryo preservation, there is still much to be learned in optimizing this protocol. Both mature and immature oocytes can be vitrified but each presents challenging aspects. Mature oocytes have microfilaments that are not yet developed in immature oocytes, which are fragile and may be disrupted by ice crystal formation during freezing. Further, currently many different cryoprotectants are used in different concentrations, most being combinations of dimethyl sulfoxide (DMSO), glycerol, and ethylene glycol. This study aimed to determine if vitrification solutions composed of ethylene glycol and either dimethyl sulfoxide or glycerol resulted in more-competent post-thaw oocytes, and to determine if maturation stage affected optimal vitrification solution. As validation of the IVF protocol, fresh mature oocytes from a commercial source were fertilized and proportion, with pronuclei formation 48 h post-IVF was recorded. Two experiments evaluated 2 cryoprotectant solutions by analysing post-vitrification and thaw competence of in vitro-fertilized oocytes to form pronuclei. Oocytes in both studies were exposed to 2 sequential vitrification solutions containing 10% DMSO or glycerol, 10% ethylene glycol and 0.5 M sucrose, and then 20% DMSO/glycerol and ethylene glycol and 0.5 M sucrose, before vitrification on cryolocks. In the first study, immature bovine oocytes (n = 200) were vitrified. Following thawing and IVM, they were analysed for pronuclei formation, with 8.49% and 0% fertilization following vitrification in DMSO and glycerol, respectively (P

Although vitrification is becoming increasingly common for oocyte preservation, there has been recent evidence that some cryoprotectants may alter DNA methylation and so result in decreased oocyte developmental competence and abnormal embryonic development post-warming. The objective of this study was to determine the effect of dimethyl sulfoxide (DMSO)- or glycerol-based vitrification protocols on DNA methylation in bovine cumulus-oocyte complexes (COC). The vitrification protocols evaluated included a combination of ethylene glycol (EG) with either DMSO or glycerol (GLY). Cumulus-oocyte complexes were first exposed to equilibration solution (ES) consisting of 7.5% DMSO or GLY and 7.5% EG for 9 min at room temperature (RT) before being transferred to vitrification solution (VS) containing 15% DMSO or GLY, 15% EG, and 0.5 M sucrose. While in VS, 3 to 4 COC were loaded into an open system vitrification device (Cryolock®, BioTech Inc.) and plunged into LN within 1 min. For warming, COC were exposed to dilution solution 1 consisting of 0.5 M sucrose (37°C) for 2.5 min and to dilution solution 2 consisting of 0.25 M sucrose for 2.5 min (RT). Base media for all solution was PBS supplemented with 20% FBS. Cumulus-oocyte complexes (n = 137) were collected from crossbred cows by ovum pick-up on 3 separate days (repetitions), with half of the oocytes each day being matured to metaphase II (MII) stage before vitrification and the other half vitrified as germinal vesicle (GV) COC. The resulting treatments were DMSO GV v. Glycerol GV and DMSO MII v. Glycerol MII. Fresh COC (GV and MII stage, respectively) were utilised as controls. DNA methylation analysis of oocytes was performed through fluorescent staining using a primary antibody anti-5 mC (1:1000) and secondary antibody Alexa Fluor 488 (1:500). Fluorescence intensity of samples was measured using ImageJ software (NIH, Bethesda, MD, USA). Data were analysed through ANOVA with post hoc Tukey’s test. No differences between the vitrification groups or controls were found when analysing DNA methylation of GV stage COC (P = 0.1825). Metaphase II stage COC showed statistical difference between groups (P

Vitrification of mammalian gametes and embryos has become the cryopreservation tool of choice in research and commercial clinical programs because of its high efficiency. Vitrification relies upon high cooling rates. In this regard the use of open system vitrification devices (OSVD) provides the highest cooling-warming rates. A limiting factor of vitrification research in domestic animals is the high cost of OSVD. Reuse of these devices could be a viable alternative for cost reduction in vitrification research projects. The objective of this study was to evaluate the effect of the repeated use of OSVD on the developmental competence of bovine cumulus-oocyte complexes (COC). A 6-treatment factorial arrangement was evaluated, where factor A was number of uses of OSVD (new, one use, and two uses) and factor B was COC type (immature, mature). Cumulus-oocyte complexes were obtained by ovum pickup from crossbred nonlactating beef cows. The vitrification procedure consisted of exposure of COC to 7.5% ethylene glycol and 7.5% dimethyl sulfoxide for 9 min. Afterward, COC were exposed to 15% ethylene glycol, 15% dimethyl sulfoxide, and 0.5 M sucrose and loaded into the tip of the OSVD (Cryolock®) with minimal volume ( 0.05). Results of the experiment shown that no differences were detected, and similar results in terms of maturation and cleavage rate were obtained when reusing OSVD.

Fungal (Penicillium chrysogenum) and yeast (Saccharomyces cerevisiae) ATP sulfurylases were shown to have very similar kinetic and chemical properties except that the fungal enzyme (a) contains a highly reactive Cys residue (SH-1) whose modification results in sigmoidal velocity curves (Renosto, F., Martin, R. L., and Segel, I. H. (1987) J. Biol. Chem. 262, 16279-16288) and (b) is allosterically inhibited by 3'-phosphoadenosine 5'-phosphosulfate (PAPS), while the yeast enzyme displays neither of these properties. The fungal enzyme subunit (64.3 kDa, 572 amino acids) is also larger than the yeast enzyme subunit (59.3 kDa, 521 amino acids). To correlate the unique allosteric properties of the fungal enzyme with specific structural features, we cloned and sequenced the ATP sulfurylase gene (aps) from P. chrysogenum. The yeast and fungal enzymes are homologous over the first 400 amino acids and contain two regions high in basic residues which are conserved in sulfurylases from Arabidopsis and the Riftia pachyptila (hydrothermal vent tube worm) chemolithotrophic symbiont. These regions may participate in forming the binding sites for MgATP2- and SO4(2-). The fungal enzyme has no sites for MgATP2- and SO4(2-). The fungal enzyme has no significant sequence homology to the yeast enzyme in the C-terminal 172 amino acids. This C-terminal region contains SH-1 (Cys-508) and has homology to MET14 (S. cerevisiae), CYSC (E. coli), and NODQ (Rhizobium meliloti), i.e. adenosine 5'-phosphosulfate (APS) kinase. The cumulative results suggest that (a) the allosteric PAPS binding site of P. chrysogenum ATP sulfurylase is located in the C-terminal domain of the protein and (b) that this domain may have evolved from APS kinase. In spite of the homology, this C-terminal region does not account for the APS kinase activity of P. chrysogenum. Fungal ATP sulfurylase has no significant homology to (or regulatory properties in common with) CYSD or CYSN, proteins reported to comprise E. coli ATP sulfurylase (Leyh, T., Vogt, T. F., and Suo, Y. (1992) J. Biol. Chem. 267, 10405-10410).

The biotransformation of 3,4-methylenedioxy-N-methylarnphetamine (MDMA) and 3,4-methylenedioxyamphetamine (MDA) was examined in the fungus Cunninghamella echinulata. In addition to the reported mammalian metabolites (MDA, 3,4-methylenedioxybenzyl methyl ketoxime, 3,4-methylenedioxybenzyl methyl ketone) and the parent substrate, there were six novel metabolites detected. N-Acetyl-3,4-methylenedioxyamphetamine (NAcMDA) was unequivocally identified and three unidentified metabolites related to NAcMDA were also detected. N-Acetyl-3,4-methylenedioxy-1-phenyl-1-hydroxy-2-aminopropane was tentatively identified as a metabolite of MDMA. The only metabolite of MDA identified was NAcMDA. Two metabolites related to MDA remain unidentified.Key words: Cunninghamella, amphetamine, biotransformation, 3,4-methylenedioxy-N-methylamphetamine, 3,4-methylenedioxyamphetamine.

The dose limiting toxicities of the short infusion trial of the dacarbazine analog, CB10-277, were nausea and vomiting which appeared to be related to the peak plasma level of the parent drug. In addition, based on mouse studies, these dose limiting toxicities occurred at a less than optimal level of the monomethyl metabolite, the presumed species required for antitumour activity. An alternative schedule that would avoid the parent drug peak plasma levels of short infusion, while possibly allowing an increase in the amount of monomethyl metabolite produced was considered. Thus, a 24 h continuous infusion schedule, repeated every 21 days was explored. Twenty-two patients received 42 courses with a dose range of 4,700-15,000 mg m-2. The dose limiting toxicity was myelosuppression (leucopenia and thrombocytopenia). Although nausea and vomiting also occurred, it was manageable with routine antiemetic therapy. Other toxicities included diarrhoea, hallucinations, malaise, muscle ache, headache and flushing and all were < or = WHO grade 2. Pharmacokinetic studies were performed with 13 courses which included all dose levels. The mean t1/2 of the parent drug was 178 min. Area under the concentration x time curve (AUC) at the highest dose for the parent drug and the monomethyl metabolite were 2,350 and 9 mM x minutes, respectively. This monomethyl metabolite AUC and the associated myelosuppression showed a more favourable comparison to the preclinical data determined in mice than the results from the short infusion trial of CB10-277. Therefore, the recommended Phase II dose and schedule of this drug was 12,000 mg m-2 given by 24 h continuous infusion.

Synthese des derives de (3-tributylstannyl)1,4-benzoquirone a partir de derives d'(4-ethynyl-4-hydroxy)cyclobut-2-enone et de methoxy tributyl stannane

Concentrations of cadmium (Cd), lead (Pb), copper (Cu), and zinc (Zn) were measured in adult barnacles (Elminius modestus Darwin) from Waitemata and Manukau Harbours in the Auckland area, New Zealand. As in studies on sediments reported in the literature, it was possible to identify areas of likely anthropogenic influence, e.g., around the Auckland Harbour Bridge for Pb, Cu, and Zn. Groups of individuals with highest concentrations for these metals showed 19.8–23.8 mg Pb kg−1, 198–266 mg Cu kg−1, and 4460–6530 mg Zn kg−1 (95% confidence limits, dry weight basis). Cd concentrations found for all barnacles from the Auckland area ranged from 0.8 to 3.1 mg kg−1. Two samples from Omaha Beach, 60 km north of Auckland, were used as a reference. Accordingly, groups of individuals with lowest concentrations for Pb, Cu, and Zn could be allocated to this site using the Student‐Newman‐Keuls Multiple Range Test (0.5–1.3 mg Pb kg−1, 8–10 mg Cu kg−1, and 144–214 mg Zn kg−1 ; 95% confidence limits). Only Cd conc...

1. Four trimethoxyamphetamine analogues were incubated with the filamentous fungus Cunninghamella echinulata. 2. 2,4,5-Trimethoxyamphetamine and 2,5-dimethoxy-4-ethoxyamphetamine were poorly metabolized by C. echinulata ATCC 9244 and C. echinulata var. elegans ATCC 9245. 2,5-Dimethoxy-4-(n)-propoxyamphetamine was mainly metabolized through N-acetylation and O-dealkylation with minor amounts of several aliphatic hydroxylation metabolites formed. 2,5-Dimethoxy-4-methylthioamphetamine was extensively metabolized to the corresponding sulphoxide. 3. 2,5-Dimethoxy-4-methylthioamphetamine metabolism was inhibited by ethanol and quinidine. Sparteine did not inhibit the formation of the sulphoxide and may have shunted the substrate through alternate metabolic pathways. 4. Incubation conditions can affect the rate and extent of fungal biotransformation of 2,5-dimethoxy-4-methylthioamphetamine, and influence dextrose utilization, ammonia formation and pH.

1. Biotransformation studies with five concentrations of racemic propranolol were conducted using the filamentous fungus Cunninghamella echinulata ATCC 9244. 2. The rate of formation and subsequent disappearance of a new major metabolite, 8-hydroxypropranolol, was dose-dependent. Desisopropylpropranolol and 4-hydroxypropranolol were also formed. 4-Hydroxypropranolol was the major fungal metabolite in earlier studies. 3. Propranolol exerted a dose-dependent response on biotransformation, fungal growth, dextrose utilization, ammonia formation and incubation broth pH. Determination of dextrose utilization and incubation broth pH would provide reliable, cost-effective and convenient alternative methods for cytotoxicological evaluation.

The metabolism of phenazopyridine was studied in the filamentous fungus Cunninghamella echinulata (synonym C. bainieri) ATCC 9244. Metabolic products were initially isolated by HPLC and TLC, with further characterization achieved by a combination of GLC, NMR, mass spectrum (MS), and GC–MS analyses. Selected samples of the incubation broths were treated with (β-glucuronidase–arylsulfatase. In addition to the reported mammalian metabolites p-aminophenol, acetaminophen, 2′-hydroxyphenazopyridine, 4′-hydroxyphenazopyridine, and parent drug, a novel metabolite was unequivocally identified as the sulfate monoester of 4′-hydroxyphenazopyridine.para-Hydroxylation of the aromatic ring with subsequent sulfoconjugation were observed as the major routes of metabolism. Ethanol, carbon monoxide, and quinidine had inhibitory effects on the C. echinulata metabolism of phenazopyridine, suppressing formation of 4′-hydroxyphenazopyridine and its sulfate monoester. Key words: Cunninghamella echinulata, biotransformation, GLC analysis of phenazopyridine, HPLC analysis of phenazopyridine, phenazopyridine, sulfate conjugate.

1. Three methoxyamphetamine analogues have been incubated with Cunninghamella echinulata under different environmental and nutrient conditions. 2. The biotransformation of 4-methoxyamphetamine was inhibited by cobalt; the carbon source and other trace metals had no effect. The rate of biotransformation of 4-methoxyamphetamine and formation of 4-hydroxyamphetamine was greater in cultures incubated on 30 degrees angle brackets rather than flat. 3. Carbon monoxide, ethanol and quinidine had a significant effect on methoxyamphetamine metabolism. 4. Metabolism was influenced by the position of the methoxy side-chain and substrate concentration. In day 7 samples the relative order for biotransformation was 3- greater than 4- greater than 2-methoxyamphetamine. 5. O-Demethylation was the major metabolic route in the biotransformation of 4-methoxyamphetamine but occurred to a lesser extent with 3-methoxyamphetamine, and was only a trace pathway with 2-methoxyamphetamine. N-acetylation was a trace pathway.

Comparative studies of oral and intravenous administration of tritiated dextran sulfate in rats showed markedly different distribution patterns. Following IV dosing about 50 per cent of the radioactivity was recovered in feces and urine within 24 h. The major portion of the recoverable dose was eliminated in the urine as dextran sulfate within 3 h after administration. In orally treated rats only about 32 per cent of the 3H was recovered in the feces and urine, the major fraction being associated with unabsorbed dextran sulfate in the feces. The remainder of the dose in both treatment groups has apparently distributed throughout the rat body with some accumulation in the liver, kidney and spleen. Consequently, the disposition of about 67 per cent or the oral dose could not be fully accounted for by these excretion routes. However, separation with Sephadex columns showed similarities in the 24 h plasma and urine profiles of the IV and orally dosed rats which suggest that while the oral dose was absorbed as dextran sulfate, it underwent rapid metabolism to small molecular weight products prior to entering the systemic circulation which were then widely distributed within the rat.

1. rac.-4-Ethoxyamphetamine was incubated with 14 different yeast and fungal microorganisms. 4-Hydroxyamphetamine was the major metabolite; traces of N-acetyl-4-ethoxyamphetamine were also detected. 2. The major fungal (Cunninghamella) metabolite of 4-propoxyamphetamine and 4-benzyloxyamphetamine was 4-hydroxyamphetamine. The major metabolites of 4-methoxyamphetamine were N-acetyl-4-methoxyamphetamine and 4-hydroxyamphetamine. 3. Acetoin derivatives were formed when alkoxyamphetamine substrates were incubated in the presence of various fungi and yeasts. 4. The findings indicate that Cunninghamella echinulata may be a useful microbial model for drug disposition and interaction studies.

The urinary elimination of 4-hydroxyamphetamine (PHA) and a series of homologous 4-alkoxy-substituted amphetamines and their metabolites was examined after single and multiple oral administration to pregnant and non-pregnant mice. The metabolic profile and extent of biotransformation in a series of alkoxy analogues were affected by the size of the alkoxy side chain, multiple dosing and pregnancy. There were increased recoveries of both the substrate and the conjugated derivative of PHA during gestation. The major metabolic routes observed were O-dealkylation, conjugation, and aliphatic hydroxylation of the propoxy side chain. There was some evidence of oxidative deamination. Pregnancy did not alter the profile of the major metabolites detected by GLC and NMR spectroscopy.

The optimization of a simple, sensitive procedure using a chick embryotoxicity screening test (CHEST) bioassay for detection of toxic compounds is presented. Dosing protocols of eggs, using several mycotoxins (aflatoxin B„ deoxynivalenol, T-2 toxin) and appropriate controls, were evaluated for embryonic sensitivity, overall practicality of the procedure, and consistency of results. It was found that both type of carrier solvent and volume injected could significantly affect overall embryonic mortality. The chick embryo was most sensitive to the effects of toxins and solvents after 1 or 2 days of incubation; a rapid decrease in response was observed as the age of the embryo at dosing increased. Following administration of the toxins just below the shell membrane by way of a small hole (

Fifty-four barrows (27.5 ± 0.5 kg) were given ad libitum for 7 wk one of 18 diets prepared from field corn inoculated with different strains of Fusarium graminearum, naturally contaminated corn and wheat, pure deoxynivalenol (DON), and clean corn. The effect of 4.7 mg pure DON kg−1 feed was not significantly different from that of the control, whereas pigs on several diets at a similar DON level had significantly lower feed intake and weight gain values. There was no correlation between DON concentration in the diets, amount ingested and pig performance. Key words: Vomitoxin, deoxynivalenol, pigs, inoculated corn, Fusarium, mycotoxins

Using a combination of radioisotopic counting and chromatographic detection techniques, the kinetics and metabolic fate of deoxynivalenol (DON) in plasma, urine and bile were studied in lactating sheep, as was the transmission of residues to milk. Following intravenous administration, the plasma clearance of 14C-DON-derived radioactivity was rapid and followed a tri-phasic decay curve comprised of a bi-exponential decrease in DON (rapid distribution phase, t1/2 alpha = 16.2 min; slower elimination phase t1/2 beta = 66.5 min) and the formation and elimination (t1/2 beta = 188.0 min) of its major plasma metabolite, DON-glucuronide conjugate, which accounted for 13% of the plasma radioactivity levels. DON was rapidly cleared from the body by metabolism to 7 possible metabolites, which were excreted essentially in the urine (91%) and to a lesser extent in the bile (6%). Most (67%) of the recovered radioactivity was in the form of the glucuronide conjugates of DON (54%) and the de-epoxide metabolite, DOM-1 (13%). Excretion of unmetabolized DON accounted for 11%. The remaining recovered dose (18%) comprised of minor amounts of DOM-1 (6%), DON-sulfate conjugate (2%) and 3 unidentified radioactive components (10%). Studies on the presence of DON-derived residues in milk indicated that, relative to the dose, only trace amounts were transmitted following either oral or iv administration of the toxin.

The technetium phosphate bone scans of 106 children with suspected septic arthritis were reviewed to determine whether the bone scan can accurately differentiate septic from nonseptic arthropathy. Only 13% of children with proved septic arthritis had correct "blind" scan interpretation. The clinically adjusted interpretation did not identify septic arthritis in 30%. Septic arthritis was incorrectly identified in 32% of children with no evidence of septic arthritis. No statistically significant differences were noted between the scan findings in the septic and nonseptic groups and no scan findings correlated specifically with the presence or absence of joint sepsis.

Diets containing deoxynivalenol (DON) prepared from Fusarium inoculated corn and Luprosil® NC, a propionate-based feed preservative, were offered to 34-kg barrows for 7 wk. The preservative improved overall performance of pigs fed the control or DON-containing diets, but did not appear to specifically reduce the adverse effects of DON. Key words: Deoxynivalenol, pigs, feed preservative, propionate, Fusarium graminearum

Cyclosporin A was produced in a low foaming semi-synthetic media by carrageenan-entrappedTolypocladium inflatum in an airlift bioreactor with an external circulation loop. The cyclosporin A recovered by a single ethyl acetate extraction was essentially free of contaminating media and endogenously produced microbial products.

Aeromonas proteolytica was grown for various time periods in nutrient broth, tryptic soy broth, a semisynthetic medium, and 1 and 5% peptone under different conditions involving temperature and in continuous shake and stationary flasks. The cell-free culture filtrates were tested for hemolytic, endopeptidase, and dermonecrotic activity and optimal growth conditions for their production were determined. The dermonecrotic activity and endopeptidase activity was found to be parallel in all tests, while hemolysin was independent of the other two. Studies on the thermal stability of the culture filtrate revealed that hemolysin and dermonecrotic and endopeptidase activity were destroyed at 70 °C for 30 min. Fractionation of the filtrate by Sephadex G-200 resolved three peaks at 280 nm. Peak I was inactive; peak II contained endopeptidase and dermonecrotic and hemolytic activity; peak III contained pigment and hemolysin. Evidence is presented that the endopeptidase and dermonecrotic substance found in the cell-free filtrates of A. proteolytica grown medium appear at the same time and thus may be the same entity.

Deprenyl and pargyline were readily metabolized by Cunninghamella echinulata ATCC 9244 and the products obtained were identified by gas-liquid chromatography and mass spectrometry. Deprenyl was completely metabolized to four products; amphetamine, N-methylamphetamine, 1-phenyl 2-propanone oxime and N-acetylamphetamine. Pargyline metabolism extracts contained substrate and five products; N-propargylbenzylamine, N-hydroxy-N-propargylbenzylamine, N-methylbenzylamine, N-acetylbenzylamine and benzylamine. Substrate concentration influenced the relative amounts of metabolites recovered. C. echinulata is an excellent organism for use as a model of mammalian metabolism.

New methods for the synthesis of functionalized alkylidene cyclopentenone and alkylidene indanone derivatives are described. The first, a method based on the reaction of cationic stoichiometric maleoylcobalt complexes with terminal alkynes, allows the synthesis of 5-alkylidene cyclopent-Z-ene-l,4-diones by reaction with terminal alkynes. The formation of the alkylidene cyclopentenedione ring system is presumed to arise via the intermediacy of a cationic vinylidene com- plex formed via tautomerization of the terminal alkyne within the coor- dination sphere of the cobalt. A more practical process for the preparation of alkylidene cyclopentenediones and 2-alkylidene indan-l- ones was developed based on the palladium(I1) induced electrophilic ring expansion of 4-(l-alkynyl)-4-hydroxycyclobut-Z-en-l-one, 2-(I- alkynyl)-2-hydroxy benzocyclobutenone, and various derivatives. Some of the compounds prepared were assayed for in vitro antitumor activity. Natural products containing or derived from an alkylidene cyclopentenone substructure show significant biological activity. For example, there is a growing class of cyclopentanoid antibiotics2 such as methylenomycin A and B,3 1 and 2, the penten~mycins,~ 3, ~anthocidin,~ - 4, and the known antitumor agent sarcomycin,6 5. eicosanoids related to the prostaglandins, SUCK as the clavulones7 (claviridenones*) 6, chloro9-bromo- and indovulones" 7, and the punaglandinsll 8, have been reported to possess remarkable cytotoxicity in both in vitro and in vivo studies.12 In fact, the non-naturally occurring arylidene cyclopentenediones 9 also show reasonable in vitro antitumor activity. l3 The significant biological activity of the alkylidene cyclopentenone natural and non-natural products has prompted the development of numerous methods for the synthesis of this class of compounds. l4 We describe herein new methods for the preparation of functionalized alkylidene cyclopentenones and alkylidene indanones. More recently, a series of marine

The major difficulty in applying high strain fatigue data to technological problems lies not in the fatigue aspect per se but in the prediction of the cyclic strain amplitudes. In this paper they are postulated for the cyclic bending of beams using large deflection theory and taking into account the changing stress-strain relationships which occur as cycling progresses. These theories have been tested using beams of rectangular cross section made of three different materials: mild steel, stainless steel, and an aluminum alloy. Good correlation has verified their applicability.

A liquid chromatographic method for any liquid mixture of the 3 herbicides 2,4-D, MCPP, and dicamba, found in commercial formulations, has been collaboratively studied. Each collaborator received 4 samples, 3 of which were ternary formulations and one a binary formulation of amine salts. Concentrations in the formulations range from 0.02 to 2.32% for dicamba, 0.32 to 21.7% for 2,4-D, and 0.22 to 11.6% for MCPP. A binary solvent system and the use of a specified 25 cm column of RP 10 μm in the reverse phase ion suppression mode will selectively quantitate each herbicide, separating many impurities found in the technical products. Standard deviations in each of 11 columns of results obtained indicate good precision. The method has been adopted official first action.

(+/-)-N-(n-propyl) amphetamine (I), a secondary amine, was readily metabolized by Cumminghamella echinulata. The products included known C- and N-oxygenated mammalian metabolites as well as N-acetylamphetamine and were identified by gas chromatography and mass spectrometry.

Amphetamine and five N-alkylated homologues were readily metabolized by Mycobacterium smegmatis and the products obtained were identified by gas–liquid chromatography and mass spectrometry. The N-alkyl substituent had a considerable influence on the degree and mechanism of biotransformation. With the exception of the N-isopropyl derivative, all of the N-alkylated homologues were dealkylated to amphetamine which was then conjugated to the N-acetyl derivative. The degree of N-oxygenation of these substrates was significantly different from that observed in mammalian and fungal systems where four products are generally recovered. Mycobacterium smegmatis N-oxygenation of amphetamine did not occur, whereas all N-alkylated amphetamines were converted to the corresponding nitrones or, in the case of methamphetamine, to 1-phenyl-2-propanone oxime. No other N-oxygenated products were isolated. Mycobacterium smegmatis metabolism of 1-phenyl-2-propanone oxime, N-hydroxyamphetamine. N-hydroxy-N-(n-propyl)amphetamine, and the nitrone, α-methyl-N-(n-propylidene) benzeneethanamine N-oxide, was also studied. Some hydrolysis of the oxime to 1-phenyl-2-propanone was observed. The other three substrates were metabolized to amphetamine and N-acetylamphetamine.

1. Incubations of phenylethylamine and 11 of its analogues with Cunninghamella bainieri have been performed. 2. Products of all major routes of mammalian metabolism including ring and aliphatic hydroxylation, oxidative deamination, N-oxidation, N-dealkylation, and conjugation were found in extracts of the fungal incubation broths. 3. Metabolism was influenced by the nature and degree of substitution.

New methods for the synthesis of functionalized alkylidene cyclopentenone and alkylidene indanone derivatives are described. The first, a method based on the reaction of cationic stoichiometric maleoylcobalt complexes with terminal alkynes, allows the synthesis of 5-alkylidene cyclopent-Z-ene-l,4-diones by reaction with terminal alkynes. The formation of the alkylidene cyclopentenedione ring system is presumed to arise via the intermediacy of a cationic vinylidene com- plex formed via tautomerization of the terminal alkyne within the coor- dination sphere of the cobalt. A more practical process for the preparation of alkylidene cyclopentenediones and 2-alkylidene indan-l- ones was developed based on the palladium(I1) induced electrophilic ring expansion of 4-(l-alkynyl)-4-hydroxycyclobut-Z-en-l-one, 2-(I- alkynyl)-2-hydroxy benzocyclobutenone, and various derivatives. Some of the compounds prepared were assayed for in vitro antitumor activity. Natural products containing or derived from an alkylidene cyclopentenone substructure show significant biological activity. For example, there is a growing class of cyclopentanoid antibiotics2 such as methylenomycin A and B,3 1 and 2, the penten~mycins,~ 3, ~anthocidin,~ - 4, and the known antitumor agent sarcomycin,6 5. eicosanoids related to the prostaglandins, SUCK as the clavulones7 (claviridenones*) 6, chloro9-bromo- and indovulones" 7, and the punaglandinsll 8, have been reported to possess remarkable cytotoxicity in both in vitro and in vivo studies.12 In fact, the non-naturally occurring arylidene cyclopentenediones 9 also show reasonable in vitro antitumor activity. l3 The significant biological activity of the alkylidene cyclopentenone natural and non-natural products has prompted the development of numerous methods for the synthesis of this class of compounds. l4 We describe herein new methods for the preparation of functionalized alkylidene cyclopentenones and alkylidene indanones. More recently, a series of marine

1. Incubations of racemic propranolol alone or in the presence of either quinidine or sparteine were performed with Cunninghamella bainieri. 2. Five mammalian metabolites of propranolol (4-hydroxypropranolol, desisopropyl-propranolol, 1-naphthoxylactic acid, propranolol glycol and 1-naphthoxyacetic acid) were present in unhydrolysed extracts of the incubation medium according to h.p.l.c. and g.l.c. analyses. The relative proportion of 4-hydroxypropranolol increased after enzymic treatment. 3. Propranolol not only had a fungistatic effect, but also caused morphological changes in the organism, which were accompanied by decomposition of 4-hydroxypropranolol and formation of a greenish-brown colour in the incubation medium. 4. Drug interaction experiments yielded results which paralleled those reported in mammals. 5. The findings indicate that C. bainieri may be a useful microbial model for drug disposition and interaction studies.

1. The fungus C. bainieri, incubated for 7 days with methoxyphenamine alone or in combination with either sparteine or quinidine, gave N-desmethylmethoxyphenamine and its N-acetyl conjugate as major metabolites, while O-desmethylmethoxyphenamine, 5-hydroxymethoxyphenamine and 2-hydroxyamphetamine were produced in lesser amounts. In addition, 1-(2-hydroxyphenyl)-2-aminopropane, 1-hydroxy-1-(2-methoxyphenyl)-2-propanone, beta-hydroxymethoxyphenamine, and 1-(5-hydroxy-2-methoxyphenyl)-2-aminopropane were tentatively identified as minor components of the fungal biotransformation of methoxyphenamine. 2. As observed in mammalian systems, the addition of either sparteine or quinidine decreased the rate and extent of methoxyphenamine biotransformation. 3. C. bainieri may be a useful model for drug interaction studies.

Rat trophoblast tissue was found to synthesize a number of low molecular weight proteins possessing prolactin-like characteristics. There appear to be at least three proteins that cross-react with antisera to pituitary prolactin. Two of the proteins had a molecular weight of 25 000, similar to ovine pituitary prolactin, and isoelectric points of 6·8 and 7·0. The third immunoreactive protein had a lower molecular weight (23 500), similar in size to human placental lactogen, and a slightly more acidic isoelectric point of 6·75. The molecular weight variants cross-reacted with an antipeptide serum that was generated to a synthetic peptide representing amino acids 150 to 164 of rat placental lactogen-2 (PL-2). Based on this analysis, we consider these proteins to be related to PL-2.Analysis of trophoblast proteins by gel-filtration chromatography resulted in the identification of another trophoblast prolactin. This material eluted earlier than PL-2-related proteins on a gel-filtration column, possessed prolactin-like activity (determined by competition with ovine pituitary prolactin for rabbit mammary gland or rat liver prolactin receptors) but showed limited cross-reactivity with either the antiserum to pituitary prolactin or the antiserum to the PL-2 peptide. We have thus identified multiple low molecular weight trophoblast prolactins, possessing different biochemical and immunological characteristics. J. Endocr. (1988) 116, 101–106