The microbial metabolism of acetophenone. Metabolism of acetophenone and some chloroacetophenones by an Arthrobacter species

1. An organism that utilizes acetophenone as sole source of carbon and energy was isolated in pure culture and tentatively identified as an Arthrobacter sp. 2. Cell-free extracts of the acetophenone-grown organism contained an enzyme, acetophenone oxygenase, that catalysed an NADPH-dependent consumption of O(2) in the presence of the growth substrate; approx. 1mol of O(2) and 1mol of NADPH were consumed per mol of acetophenone oxidized. 3. Cell-free extracts also contained an enzyme capable of the hydrolysis of phenyl acetate to phenol and acetate. The amount of this esterase was increased markedly by growth on acetophenone. 4. The observed products of the acetophenone oxygenase reaction by crude cell-free extracts were phenol and acetate. However, inhibition of the phenyl acetate esterase by paraoxon resulted in the formation of phenyl acetate from acetophenone. 5. A degradative sequence is proposed in which acetophenone is metabolized by an oxygen-insertion reaction to form phenyl acetate. Further metabolism occurs by hydrolysis of this ester. 6. The organism and extracts were shown to metabolize chlorinated acetophenones. The environmental implications of this observation are discussed.     

Clinical and immunological responses in metastatic melanoma patients vaccinated with a high-dose poly-epitope vaccine

Background

Safety and cellular immunogenicity of rising doses and varying regimens of a poly-epitope vaccine were evaluated in advanced metastatic melanoma. The vaccine comprised plasmid DNA and recombinant modified vaccinia virus Ankara (MVA) both expressing a string (Mel3) of seven HLA.A2/A1 epitopes from five melanoma antigens.

Methods

Forty-one HLA-A2 positive patients with stage III/IV melanoma were enrolled. Patient groups received one or two doses of DNA.Mel3 followed by escalating doses of MVA.Mel3. Immunisations then continued eight weekly in the absence of disease progression. Epitope-specific CD8+ T cell responses were evaluated using ex-vivo tetramer and IFN-γ ELISPOT assays. Safety and clinical responses were monitored.

Results

Prime-boost DNA/MVA induced Melan-A-specific CD8+ T cell responses in 22/31 (71%) patients detected by tetramer assay. ELISPOT detected a response to at least one epitope in 10/31 (32%) patients. T cell responder rates were <50% with low-dose DNA/MVA, or MVA alone, rising to 91% with high-dose DNA/MVA. Among eight patients showing evidence of clinical benefit—one PR (24 months+), five SD (5 months+) and two mixed responses—seven had associated immune responses. Melan-A-tetramer+ immunity was associated with a median 8-week increase in time-to-progression (P = 0.037) and 71 week increase in survival (P = 0.0002) compared to non-immunity. High-dose vaccine was well tolerated. The only significant toxicities were flu-like symptoms and injection-site reactions.

Conclusions

DNA.Mel3 and MVA.Mel3 in a prime-boost protocol generated high rates of immune response to melanoma antigen epitopes. The treatment was well tolerated and the correlation of immune responses with patient outcomes encourages further investigation.     

N-(Pyridin-2-yl) arylsulfonamide inhibitors of 11β-hydroxysteroid dehydrogenase type 1: Strategies to eliminate reactive metabolites

N-(Pyridin-2-yl) arylsulfonamides 1 and 2 (PF-915275) were identified as potent inhibitors of 11β-hydroxysteroid dehydrogenase type 1. A screen for bioactivation revealed that these compounds formed glutathione conjugates. This communication presents the results of a risk benefit analysis carried out to progress 2 (PF-915275) to a clinical study and the strategies used to eliminate reactive metabolites in this series of inhibitors. Based on the proposed mechanism of bioactivation and structure–activity relationships, design efforts led to N-(pyridin-2-yl) arylsulfonamides such as 18 and 20 that maintained potent 11β-hydroxysteroid dehydrogenase type 1 activity, showed exquisite pharmacokinetic profiles, and were negative in the reactive metabolite assay.

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Effects of Cortisol and Dexamethasone on Insulin Signalling Pathways in Skeletal Muscle of the Ovine Fetus during Late Gestation

Before birth, glucocorticoids retard growth, although the extent to which this is mediated by changes in insulin signalling pathways in the skeletal muscle of the fetus is unknown. The current study determined the effects of endogenous and synthetic glucocorticoid exposure on insulin signalling proteins in skeletal muscle of fetal sheep during late gestation. Experimental manipulation of fetal plasma glucocorticoid concentration was achieved by fetal cortisol infusion and maternal dexamethasone treatment. Cortisol infusion significantly increased muscle protein levels of Akt2 and phosphorylated Akt at Ser473, and decreased protein levels of phosphorylated forms of mTOR at Ser2448 and S6K at Thr389. Muscle GLUT4 protein expression was significantly higher in fetuses whose mothers were treated with dexamethasone compared to those treated with saline. There were no significant effects of glucocorticoid exposure on muscle protein abundance of IR-β, IGF-1R, PKCζ, Akt1, calpastatin or muscle glycogen content. The present study demonstrated that components of the insulin signalling pathway in skeletal muscle of the ovine fetus are influenced differentially by naturally occurring and synthetic glucocorticoids. These findings may provide a mechanism by which elevated concentrations of endogenous glucocorticoids retard fetal growth.     

Factors affecting 'Hass' avocado fruit size: Carbohydrate, abscisic acid and isoprenoid metabolism in normal and phenotypically small fruit

Carbohydrate and abscisic acid (ABA) metabolism were investigated in normal and phenotypically small 'Hass' avocado ( Persea americana Mill.) fruit in an attempt to link alterations in sugar and ABA content with changes in 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR, EC 1.1.1.34) activity and fruit size. The small-fruit phenotype was characterized by reduced seed HMGR activity, increased seed insoluble acid invertase ( β - d -fructofuranosidase, EC 3.2.1.26), decreased sucrose synthase (SS; UDP- d -glucose: d -fructose-2- α -glucosyl-transferase, EC 2.4.1.13) activity, decreased sucrose content, and increased glucose as a proportion of the total soluble sugar. Sucrose phosphate synthase (SPS; UDP- d -glucose: d -fructose 6-phosphate 2- α - d -glucosyltransferase, EC 2.4.1.14) activity was unaffected in seed but reduced in mesocarp of the small fruit. In addition, the small-fruit variant displayed enhanced respiration and both seed and mesocarp tissue showed increased ABA metabolism. Applied ABA caused an increase in insoluble acid invertase activity in seed tissue of normal fruit while mevastatin reduced HMGR activity in this tissue, caused sucrose depletion and increased the proportion of glucose from 5 to 57% of total soluble sugars. Exogenous glucose suppressed HMGR activity in seed tissue whereas in mesocarp tissue, HMGR activity was reduced to 38% of the control after 6 h but enhanced by 46% by 48 h. Glucose increased ABA biosynthesis and turnover in competent tissues. These results suggest that ABA turnover is mediated, in part, by carbohydrate content and composition which also affects HMGR activity. It is proposed that sugar and ABA signals act in concert to modulate expression and/or activity of HMGR in the control of 'Hass' avocado fruit growth and final fruit size.     

Assembly of thick filaments and myofibrils occurs in the absence of the myosin head

We investigated the importance of the myosin head in thick filament formation and myofibrillogenesis by generating transgenic Drosophila lines expressing either an embryonic or an adult isoform of the myosin rod in their indirect flight muscles. The headless myosin molecules retain the regulatory light-chain binding site, the alpha-helical rod and the C-terminal tailpiece. Both isoforms of headless myosin co-assemble with endogenous full-length myosin in wild-type muscle cells. However, rod polypeptides interfere with muscle function and cause a flightless phenotype. Electron microscopy demonstrates that this results from an antimorphic effect upon myofibril assembly. Thick filaments assemble when the myosin rod is expressed in mutant indirect flight muscles where no full-length myosin heavy chain is produced. These filaments show the characteristic hollow cross-section observed in wild type. The headless thick filaments can assemble with thin filaments into hexagonally packed arrays resembling normal myofibrils. However, thick filament length as well as sarcomere length and myofibril shape are abnormal. Therefore, thick filament assembly and many aspects of myofibrillogenesis are independent of the myosin head and these processes are regulated by the myosin rod and tailpiece. However, interaction of the myosin head with other myofibrillar components is necessary for defining filament length and myofibril dimensions.     

Bacterial otitis media: current vaccine development strategies

Otitis media is the most common reason for children less than 5 years of age to visit a medical practitioner. Whilst the disease rarely results in death, there is significant associated morbidity. The most common complication is loss of hearing at a critical stage of the development of speech, language and cognitive abilities in children. The cause and pathogenesis of otitis media is multifactorial. Among the contributing factors, the single most important are viral and bacterial infections. Infection with respiratory syncytial virus, influenza viruses, para-influenza viruses, enteroviruses and adenovirus are most commonly associated with acute and chronic otitis media. Streptococcus pneumoniae, non-typeable Haemophilus influenzae and Moraxella catarrhalis are the most commonly isolated bacteria from the middle ears of children with otitis media. Treatment of otitis media has largely relied on the administration of antimicrobials and surgical intervention. However, attention has recently focused on the development of a vaccine. For a vaccine to be effective against bacterial otitis media, it must, at the very least, contain antigens that induce a protective immune response in the middle ear against the three most common infecting bacteria. Whilst over the past decade there has been significant progress in the development of vaccines against invasive S. pneumoniae disease, these vaccines are less efficacious for otitis media. The search for candidate vaccine antigens for non-typeable H. influenzae are well advanced whilst less progress has been made for M. catarrhalis. No human studies have been conducted for non-typeable H. influenzae or M. catarrhalis and the concept of a tribacterial vaccine remains to be tested in animal models. Only when vaccine antigens are determined and an understanding of the immune responses induced in the middle ear by infection and immunization is gained will the formulation of a tribacterial vaccine against otitis media be possible.     

Influence of insects and fungal pathogens on individual and population parameters of <Emphasis Type="Italic">Cirsium arvense</Emphasis> in its native and introduced ranges

Introduced weeds are hypothesized to be invasive in their exotic ranges due to release from natural enemies. Cirsium arvense (Californian, Canada, or creeping thistle) is a weed of Eurasian origin that was inadvertently introduced to New Zealand (NZ), where it is presently one of the worst invasive weeds. We tested the ‘enemy release hypothesis’ (ERH) by establishing natural enemy exclusion plots in both the native (Europe) and introduced (NZ) ranges of C. arvense. We followed the development and fate of individually labelled shoots and recorded recruitment of new shoots into the population over two years. Natural enemy exclusion had minimal impact on shoot height and relative growth rate in either range. However, natural enemies did have a significant effect on shoot population growth and development in the native range, supporting the ERH. In year one, exclusion of insect herbivores increased mean population growth by 2.1–3.6 shoots m⁻², and in year two exclusion of pathogens increased mean population growth by 2.7–4.1 shoots m⁻². Exclusion of insect herbivores in the native range also increased the probability of shoots developing from the budding to the reproductive growth stage by 4.0× in the first year, and 13.4× in the second year; but exclusion of pathogens had no effect on shoot development in either year. In accordance with the ERH, exclusion of insect herbivores and pathogens did not benefit shoot development or population growth in the introduced range. In either range, we found no evidence for an additive benefit of dual exclusion of insects and pathogens, and in no case was there an interaction between insect and pathogen exclusion. This study further demonstrates the value of conducting manipulative experiments in the native and introduced ranges of an invasive plant to elucidate invasion mechanisms.