Changes to insulin sensitivity in glucose clearance systems and redox following dietary supplementation with a novel cysteine-rich protein: A pilot randomized controlled trial in humans with type-2 diabetes.

We recently developed a novel keratin-derived protein (KDP) rich in cysteine, glycine, and arginine, with the potential to alter tissue redox status and insulin sensitivity. The KDP was tested in 35 human adults with type-2 diabetes mellitus (T2DM) in a 14-wk randomised controlled pilot trial comprising three 2×20 g supplemental protein/day arms: KDP-whey (KDPWHE), whey (WHEY), non-protein isocaloric control (CON), with standardised exercise. Outcomes were measured morning fasted and following insulin-stimulation (80 mU/m2/min hyperinsulinaemic-isoglycaemic clamp). With KDPWHE supplementation there was good and very-good evidence for moderate-sized increases in insulin-stimulated glucose clearance rate (GCR; 26%; 90% confidence limits, CL 2%, 49%) and skeletal-muscle microvascular blood flow (46%; 16%, 83%), respectively, and good evidence for increased insulin-stimulated sarcoplasmic GLUT4 translocation (18%; 0%, 39%) vs CON. In contrast, WHEY did not effect GCR (-2%; -25%, 21%) and attenuated HbA1c lowering (14%; 5%, 24%) vs CON. KDPWHE effects on basal glutathione in erythrocytes and skeletal muscle were unclear, but in muscle there was very-good evidence for large increases in oxidised peroxiredoxin isoform 2 (oxiPRX2) (19%; 2.2%, 35%) and good evidence for lower GPx1 concentrations (-40%; -4.3%, -63%) vs CON; insulin stimulation, however, attenuated the basal oxiPRX2 response (4%; -16%, 24%), and increased GPx1 (39%; -5%, 101%) and SOD1 (26%; -3%, 60%) protein expression. Effects of KDPWHE on oxiPRX3 and NRF2 content, phosphorylation of capillary eNOS and insulin-signalling proteins upstream of GLUT4 translocation AktSer437 and AS160Thr642 were inconclusive, but there was good evidence for increased IRSSer312 (41%; 3%, 95%), insulin-stimulated NFκB-DNA binding (46%; 3.4%, 105%), and basal PAK-1Thr423/2Thr402 phosphorylation (143%; 66%, 257%) vs WHEY. Our findings provide good evidence to suggest that dietary supplementation with a novel edible keratin protein in humans with T2DM may increase glucose clearance and modify skeletal-muscle tissue redox and insulin sensitivity within systems involving peroxiredoxins, antioxidant expression, and glucose uptake.

Optimization of the Promega PowerSeq™ Auto/Y system for efficient integration within a forensic DNA laboratory.

The application of massively parallel sequencing (MPS) is growing in the forensic DNA field, as forensic DNA laboratories are continuously seeking methods to gain information from a limited or degraded forensic sample. However, the laborious nature of current MPS methodologies required for successful library preparation and sequencing leave opportunities for improvement to make MPS a practical option for processing forensic casework. In this study, the Promega PowerSeq™ Auto/Y System Prototype, a MPS laboratory workflow that incorporates multiplex amplification, was selected for optimization with the objectives to introduce automation for relieving manual processing, and to reduce the number of steps recommended by the standard protocol. Successful changes in the optimized workflow included a switch from column-based PCR purification to automatable bead-based purification, adoption of the library preparation procedures by a liquid handling robot platform, and removal of various time-consuming quality checks. All data in this study were found to be concordant with capillary electrophoresis (CE) data and previously-generated MPS results from this workflow. Read abundance and allele balance, metrics related to sample interpretation reliability, were not significantly different when compared to samples processed with the manufacturer's protocol. All the modifications implemented resulted in increased laboratory efficiency, reduced the protocol steps associated with risk of contamination and human error events, and decreased manual processing time by approximately 12h. These findings provide forensic DNA laboratories a more streamlined option when considering implementation of a MPS workflow.

Relationships between rapid changes in local aromatase activity and estradiol concentrations in male and female quail brain.

Estradiol-17ß (E2) synthesized in the brain plays a critical role in the activation of sexual behavior in many vertebrate species. Because E2 concentrations depend on aromatization of testosterone, changes in aromatase enzymatic activity (AA) are often utilized as a proxy to describe E2 concentrations. Utilizing two types of stimuli (sexual interactions and acute restraint stress) that have been demonstrated to reliably alter AA within minutes in opposite directions (sexual interactions=decrease, stress=increase), we tested in Japanese quail whether rapid changes in AA are paralleled by changes in E2 concentrations in discrete brain areas. In males, E2 in the pooled medial preoptic nucleus/medial portion of the bed nucleus of the stria terminalis (POM/BST) positively correlated with AA following sexual interactions. However, following acute stress, E2 decreased significantly (approximately 2-fold) in the male POM/BST despite a significant increase in AA. In females, AA positively correlated with E2 in both the POM/BST and mediobasal hypothalamus supporting a role for local, as opposed to ovarian, production regulating brain E2 concentrations. In addition, correlations of individual E2 in POM/BST and measurements of female sexual behavior suggested a role for local E2 synthesis in female receptivity. These data demonstrate that local E2 in the male brain changes in response to stimuli on a time course suggestive of potential non-genomic effects on brain and behavior. Overall, this study highlights the complex mechanisms regulating local E2 concentrations including rapid stimulus-driven changes in production and stress-induced changes in catabolism.

Rapid control of reproductive behaviour by locally synthesised oestrogens: focus on aromatase.

Oestrogens activate nucleus- and membrane-initiated signalling. Nucleus-initiated events control a wide array of physiological and behavioural responses. These effects generally take place within relatively long periods of time (several hours to days). By contrast, membrane-initiated signalling affects a multitude of cellular functions in a much shorter timeframe (seconds to minutes). However, much less is known about their functional significance. Furthermore, the origin of the oestrogens able to trigger these acute effects is rarely examined. Finally, these two distinct types of oestrogenic actions have often been studied independently such that we do not exactly know how they cooperate to control the same response. The present review presents a synthesis of recent work carried out in our laboratory that aimed to address these issues in the context of the study of male sexual behaviour in Japanese quail, which is a considered as a suitable species for tackling these issues. The first section presents data indicating that 17ß-oestradiol, or its membrane impermeable analogues, acutely enhances measures of male sexual motivation but does not affect copulatory behaviour. These effects depend on the activation of membrane-initiated events and local oestrogen production. The second part of this review discusses the regulation of brain oestrogen synthesis through post-translational modifications of the enzyme aromatase. Initially discovered in vitro, these rapid and reversible enzymatic modulations occur in vivo following variations in the social and environment context and therefore provide a mechanism of acute regulation of local oestrogen provision with a spatial and time resolution compatible with the rapid effects observed on male sexual behaviour. Finally, we discuss how these distinct modes of oestrogenic action (membrane- versus nucleus-initiated) acting in different time frames (short- versus long-term) interact to control different components (motivation versus performance) of the same behavioural response and improve reproductive fitness.

Neurochemical control of rapid stress-induced changes in brain aromatase activity.

In the male brain, the medial preoptic nucleus (POM) is known to be a critical relay for the activation of sexual behaviour, with the aromatisation of testosterone into 17ß-oestradiol (E2 ) playing a key role. Acute stress has been shown to differentially modulate the aromatase enzyme in this and other brain nuclei in a sex-specific manner. In POM specifically, stress induces increases in aromatase activity (AA) that are both rapid and reversible. How the physiological processes initiated during an acute stress response mediate sex- and nuclei- specific changes in AA and which stress response hormones are involved remains to be determined. By examining the relative effects of corticosterone (CORT), arginine vasotocin (AVT, the avian homologue to arginine vasopressin) and corticotrophin-releasing factor (CRF), the present study aimed to define the hormone profile regulating stress-induced increases in AA in the POM. We found that CORT, AVT and CRF all appear to play some role in these changes in the male brain. In addition, these effects occur in a targeted manner, such that modulation of the enzyme by these hormones only occurs in the POM rather than in all aromatase-expressing nuclei. Similarly, in the female brain, the experimental effects were restricted to the POM but only CRF was capable of inducing the stress-like increases in AA. These data further demonstrate the high degree of specificity (nuclei-, sex- and hormone-specific effects) in this system, highlighting the complexity of the stress-aromatase link and suggesting modes through which the nongenomic modulation of this enzyme can result in targeted, rapid changes in local oestrogen concentrations.

Brain aromatase and circulating corticosterone are rapidly regulated by combined acute stress and sexual interaction in a sex-specific manner.

Neural production of 17ß-oestradiol via aromatisation of testosterone may play a critical role in rapid, nongenomic regulation of physiological and behavioural processes. In brain nuclei implicated in the control of sexual behaviour, sexual or stressfull stimuli induce, respectively, a rapid inhibition or increase in preoptic aromatase activity (AA). In the present study, we tested quail that were either nonstressed or acutely stressed (15 min of restraint) immediately before sexual interaction (5 min) with stressed or nonstressed partners. We measured nuclei-specific AA changes, corresponding behavioural output, fertilisation rates and corticosterone (CORT) concentrations. In males, sexual interaction rapidly reversed stress-induced increases of AA in the medial preoptic nucleus (POM). This time scale (< 5 min) highlights the dynamic potential of the aromatase system to integrate input from stimuli that drive AA in opposing directions. Moreover, acute stress had minimal effects on male behaviour, suggesting that the input from the sexual stimuli on POM AA may actively preserve sexual behaviour despite stress exposure. We also found distinct sex differences in contextual physiological responses: males did not show any effect of partner status, whereas females responded to both their stress exposure and the male partner's stress exposure at the level of circulating CORT and AA. In addition, fertilisation rates and female CORT correlated with the male partner's exhibition of sexually aggressive behaviour, suggesting that female perception of the male can affect their physiology as much as direct stress. Overall, male reproduction appears relatively simple: sexual stimuli, irrespective of stress, drives major neural changes including rapid reversal of stress-induced changes of AA. By contrast, female reproduction appears more nuanced and context specific, with subjects responding physiologically and behaviourally to stress, the male partner's stress exposure, and female-directed male behaviour.

Environmental and not maternal effects determine variation in offspring phenotypes in a passerine bird.

Maternal and environmental effects can profoundly influence offspring phenotypes, independent of genetic effects. Within avian broods, both the asymmetric post-hatching environment created by hatching asynchrony and the differential maternal investment through the laying sequence have important consequences for individual nestlings in terms of the allocation of resources to body structures with different contributions to fitness. The purpose of this study was to evaluate the relative importance of post-hatching environmental and maternal effects in generating variation in offspring phenotypes. First, an observational study showed that within blue tit, Cyanistes caeruleus, broods, late-hatched nestlings allocated resources to tarsus development, maintained mass gain and head-bill growth and directed resources away from the development of fourth primary feathers. Second, a hatching order manipulation experiment resulted in nestlings from first-laid eggs hatching last, thereby allowing comparison with both late and early-hatched nestlings. Experimental nestlings had growth patterns which were closer to late-hatched nestlings, suggesting that within-brood growth patterns are determined by post-hatching environmental effects. Therefore, we conclude that post-hatching environmental effects play an important role in generating variation in offspring phenotypes.

Chronic stress alters glucocorticoid receptor and mineralocorticoid receptor mRNA expression in the European starling (Sturnus vulgaris) brain.

Although the glucocorticoid response to acute short-term stress is an adaptive physiological mechanism that aids in the response to and survival of noxious stimuli, chronic stress is associated with a negative impact on health. In wild-caught European starlings (Sturnus vulgaris), chronic stress alters the responsiveness of hypothalamic-pituitary-adrenal (HPA) axis as measured by the acute corticosterone response. In the present study, we investigated potential underlying neuroendocrine mechanisms by comparing glucocorticoid receptor and mineralocorticoid receptor mRNA expression in the brains of chronically and nonchronically-stressed starlings. Hypothalamic paraventricular nucleus, but not hippocampal, glucocorticoid receptor mRNA expression in chronically-stressed birds was significantly lower compared to controls, suggesting changes in the efficacy of corticosterone negative feedback. In addition, chronically-stressed birds showed a significant decrease in hippocampal MR mRNA expression. Together, these results suggest that chronic stress changes the brain physiology of wild birds and provides important information for the understanding of the underlying mechanisms that result in dysregulation of the HPA axis in wild animals by chronic stress.

Expression and function of growth differentiation factor-9 in an oviparous species, Gallus domesticus.

Many studies have indicated a critical role for the oocyte growth factor, growth differentiation factor-9 (GDF9), in mammalian follicle development, but no information has been available concerning oviparous species. We cloned a cDNA for chicken GDF9 (162 base pairs) and used it in Northern blot analysis to identify a transcript at 1.7 kilobase in RNA isolated from the ovary of the hen. We also sequenced two full-length clones from a normalized chicken reproductive tract cDNA library. The cDNA clone for chicken GDF9 encodes a protein of approximately 449 amino acids and all six cysteine residues, and three of the four glycosylation sites are conserved with respect to mammalian GDF9. Chicken GDF9 is approximately 65% similar in the full-length cDNA sequence and 80% similar in amino acid sequence at the C-terminal region to GDF9 from several mammals. Quantitative polymerase chain reaction analysis (n = 5) indicated that GDF9 mRNA is greatest in follicles < 1 mm in size compared with larger follicles or granulosa layers isolated from larger follicles. Immunocytochemical analysis showed strong expression of GDF9 in hen oocytes. In yolk-filled oocytes, the GDF9 was localized at the periphery of the oocyte. Finally, oocyte-conditioned medium (from < 1-mm oocytes) resulted in a 2-fold increase in granulosa cell proliferation, which could be inhibited by preincubation of the conditioned medium with GDF9 antibody. These data suggest that GDF9 is present in the hen oocyte and that this factor is capable of enhancing granulosa cell proliferation, as has been demonstrated in mammals.

c-Jun N-terminal kinase-interacting protein 1 inhibits gene expression in response to hypertrophic agonists in neonatal rat ventricular myocytes.

G(q)-coupled receptor agonists, such as endothelin-1 (ET-1) and phenylephrine (PE), initiate a hypertrophic response in cardiac myocytes that is characterized by increased expression of atrial natriuretic factor (ANF), beta-myosin heavy chain (beta-MHC), skeletal muscle alpha-actin (SkalphaA) and ventricular myosin light chain-2 (vMLC2). ET-1 and PE activate both the extracellular signal-regulated kinases and c-Jun N-terminal kinases (JNKs) in cardiac myocytes, but the extent to which each contributes to the hypertrophic response is uncertain. Here we have used the JNK-binding domain of JNK-interacting protein 1 (JIP-1), a cytosolic scaffold protein that binds to JNK and inhibits its signalling when overexpressed, to assess the contribution of JNK activation to the hypertrophic response. Expression of JIP-1 inhibited the increase in ANF, beta-MHC, SkalphaA and vMLC2 reporter gene expression in response to ET-1 (by 45-86%) and PE (by 56-60%). However, activation of these reporter genes by PMA, which does not activate JNK significantly in myocytes, was much less affected by overexpression of JIP-1. JIP-1 also failed to inhibit reporter gene activation in response to constitutively active Ras or Raf, but attenuated reporter gene activation induced by a constitutively active mutant of mitogen-activated protein kinase kinase kinase 1 (MEKK1), an upstream kinase that preferentially activates JNKs, by 50%. Overexpression of JIP-1 also significantly reduced the increase in cell area in response to PE from 63% to 56%, but had no effect on the increase in cell size in response to ET-1 (38%). These results suggest that activation of the JNK pathway contributes to the transcriptional and morphological responses to G(q) receptor-coupled hypertrophic agonists.

Inhibition of JNK by overexpression of the JNL binding domain of JIP-1 prevents apoptosis in sympathetic neurons.

Studies in non-neuronal cells show that c-Jun N-terminal kinases (JNK) play a key role in apoptotic cell death. In some neurons JNK is also thought to initiate cell death by the activation of c-Jun. JNK inhibition has been achieved pharmacologically by inhibiting upstream kinases, but there has been no direct demonstration that inhibition of JNK can prevent neuronal death. We have therefore examined whether the JNK binding domain (JBD) of JNK-interacting protein-1 (JIP-1, a scaffold protein and specific inhibitor of JNK) can inhibit c-Jun phosphorylation and support the survival of sympathetic neurons deprived of NGF. We show that expression of the JBD in >80% of neurons was sufficient to prevent the phosphorylation of c-Jun and its nuclear accumulation as well as abrogate neuronal cell death induced by NGF deprivation. JBD expression also preserved the capacity of mitochondria to reduce MTT. Interestingly, although the PTB domain of JIP was reported to interact with rhoGEF, expression of the JBD domain was sufficient to localize the protein to the membrane cortex and growth cones. Hence, JNK activation is a key event in apoptotic death induced by NGF withdrawal, where its point of action lies upstream of mitochondrial dysfunction.

Dual roles for c-Jun N-terminal kinase in developmental and stress responses in cerebellar granule neurons.

c-Jun N-terminal kinases (JNKs) typically respond strongly to stress, are implicated in brain development, and are believed to mediate neuronal apoptosis. Surprisingly, however, JNK does not respond characteristically to stress in cultured cerebellar granule (CBG) neurons, a widely exploited CNS model for studies of death and development, despite the regulation of its substrate c-Jun. To understand this anomaly, we characterized JNK regulation in CBG neurons. We find that the specific activity of CBG JNK is elevated considerably above that from neuron-like cell lines (SH-SY5Y, PC12); however, similar elevated activities are found in brain extracts. This activity does not result from cellular stress because the stress-activated protein kinase p38 is not activated. We identify a minor stress-sensitive pool of JNK that translocates with mitogen-activated protein kinase kinase-4 (MKK4) into the nucleus. However, the major pool of total activity is cytoplasmic, residing largely in the neurites, suggesting a non-nuclear role for JNK in neurons. A third JNK pool is colocalized with MKK7 in the nucleus, and specific activities of both increase during neuritogenesis, nuclear JNK activity increasing 10-fold, whereas c-Jun expression and activity decrease. A role for JNK during differentiation is supported by modulation of neuritic architecture after expression of dominant inhibitory regulators of the JNK pathway. Channeling of JNK signaling away from c-Jun during differentiation is consistent with the presence in the nucleus of the JNK/MKK7 scaffold protein JNK-interacting protein, which inhibits JNK-c-Jun interaction. We propose a model in which distinct pools of JNK serve different functions, providing a basis for understanding multifunctional JNK signaling in differentiating neurons.

JNK (c-Jun N-terminal kinase) and p38 activation in receptor-mediated and chemically-induced apoptosis of T-cells: differential requirements for caspase activation.

Activation of the stress-activated mitogen-activated protein kinases (MAP kinases), c-Jun N-terminal kinase (JNK) and p38, is necessary for the induction of apoptosis in neuronal cells; however, in other cell types their involvement may be stimulus-dependent. In the present study we investigate the activation of JNK and p38 in a single non-neuronal cell type, undergoing receptor-mediated (tumour necrosis factor-related apoptosis-inducing ligand and CD95) or chemically-induced (lactacystin) apoptosis. In Jurkat T-cells, receptor-mediated and chemically-induced apoptosis resulted in a time-dependent activation of the initiator caspases-8 and -9, respectively. Both types of stimuli resulted in a significant activation of JNK and p38, which closely paralleled the time-dependent induction of apoptosis. The caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp-(OMe) fluoromethyl ketone (z-VAD.FMK) inhibited receptor-mediated apoptosis and suppressed JNK and p38 activation. In contrast, inhibition of lactacystin-induced apoptosis with z-VAD.FMK, as assessed by phosphatidylserine exposure and poly(ADP-ribose) polymerase cleavage, did not inhibit activation of JNK or p38, demonstrating that during chemically-induced apoptosis, activation of JNK and p38 is independent of effector caspases. The role of p38 in apoptosis was assessed using the specific p38 inhibitor, SB203580. No effect on the induction of apoptosis or caspase activation was observed, although activation of mitogen-activated protein kinase-activated protein kinase-2 (MAPKAPK-2), an immediate downstream target of p38, was inhibited. Therefore neither p38 activation nor activation of MAPKAPK-2 is critical for induction of either receptor- or chemically-induced apoptosis. Thus, within a single cell type, (1) the mechanism of p38 and JNK activation during apoptosis is stimulus-dependent and (2) activation of the p38 pathway is not required for caspase activation or apoptosis, assessed by phosphatidylserine exposure, but may still be required to elicit other features of the apoptotic phenotype.

c-Myc protein synthesis is initiated from the internal ribosome entry segment during apoptosis.

Recent studies have shown that during apoptosis protein synthesis is inhibited and that this is in part due to the proteolytic cleavage of eukaryotic initiation factor 4G (eIF4G). Initiation of translation can occur either by a cap-dependent mechanism or by internal ribosome entry. The latter mechanism is dependent on a complex structural element located in the 5' untranslated region of the mRNA which is termed an internal ribosome entry segment (IRES). In general, IRES-mediated translation does not require eIF4E or full-length eIF4G. In order to investigate whether cap-dependent and cap-independent translation are reduced during apoptosis, we examined the expression of c-Myc during this process, since we have shown previously that the 5' untranslated region of the c-myc proto-oncogene contains an IRES. c-Myc expression was determined in HeLa cells during apoptosis induced by tumor necrosis factor-related apoptosis-inducing ligand. We have demonstrated that the c-Myc protein is still expressed when more than 90% of the cells are apoptotic. The presence of the protein in apoptotic cells does not result from either an increase in protein stability or an increase in expression of c-myc mRNA. Furthermore, we show that during apoptosis initiation of c-myc translation occurs by internal ribosome entry. We have investigated the signaling pathways that are involved in this response, and cotransfection with plasmids which harbor either wild-type or constitutively active MKK6, a specific immediate upstream activator of p38 mitogen-activated protein kinase (MAPK), increases IRES-mediated translation. In addition, the c-myc IRES is inhibited by SB203580, a specific inhibitor of p38 MAPK. Our data, therefore, strongly suggest that the initiation of translation via the c-myc IRES during apoptosis is mediated by the p38 MAPK pathway.

Interaction of a mitogen-activated protein kinase signaling module with the neuronal protein JIP3.

The c-Jun NH(2)-terminal kinase (JNK) group of mitogen-activated protein kinases (MAPKs) is activated in response to the treatment of cells with inflammatory cytokines and by exposure to environmental stress. JNK activation is mediated by a protein kinase cascade composed of a MAPK kinase and a MAPK kinase kinase. Here we describe the molecular cloning of a putative molecular scaffold protein, JIP3, that binds the protein kinase components of a JNK signaling module and facilitates JNK activation in cultured cells. JIP3 is expressed in the brain and at lower levels in the heart and other tissues. Immunofluorescence analysis demonstrated that JIP3 was present in the cytoplasm and accumulated in the growth cones of developing neurites. JIP3 is a member of a novel class of putative MAPK scaffold proteins that may regulate signal transduction by the JNK pathway.

Increased, not decreased activation of the insulin-like growth factor (IGF) receptor signalling pathway during ceramide-induced apoptosis.

The insulin-like growth factors (IGFs) are capable of blocking apoptosis in many cell lines in vitro. The IGF-I receptor (IGF-IR) is believed to mediate protective effects of the IGFs against apoptosis. To determine whether ceramide-mediated induction of apoptosis involved a decreased survival effect of the IGF-IR, apoptosis was induced in IGF-I receptor positive (R+) and negative (R-) murine fibroblasts by incubation with increasing doses of the sphingolipid analogue, C2 ceramide. Lower ceramide doses were required to induce death in receptor negative compared with receptor positive fibroblasts (P< 0.05 at ceramide doses of 2 microM or greater), not only corroborating evidence that the IGF-I receptor functions as a survival receptor, but also suggesting that ceramide is not inducing apoptosis by suppressing a survival effect of the IGF-IR. Ceramide has been reported to induce death through suppression of MAP kinase, and activation of JUN kinase signalling; since our initial data suggested that ceramide had not affected an anti-apoptotic signalling event of the IGF-IR, we monitored the activation of these enzymes. To our surprise, in the presence of ceramide, not only was JUN kinase activity increased, but so too was MAP kinase. Inhibition of MAP kinase, using the MEKK inhibitor, PD98059, significantly reduced ceramide-induced cell death (P< 0. 001). Ceramide also enhanced IGF-induced tyrosine phosphorylation of the IGF-I receptor and activated PI-3 kinase. The cumulative effects of these events resulted in increased progression to the G2 phase of the cell cycle, arrest without subsequent mitosis, and apoptosis. These results indicate that ceramide is capable of eliciting apparently contradictory events within a single cell type, and suggest that in the presence of an IGF-IR, survival is enhanced because ceramide can activate PI-3 kinase, believed to be an anti-apoptotic enzyme.

Purification, properties, and characterization of recombinant Streptomyces sp. strain C5 DoxA, a cytochrome P-450 catalyzing multiple steps in doxorubicin biosynthesis.

DoxA is a cytochrome P-450 monooxygenase involved in the late stages of daunorubicin and doxorubicin biosynthesis that has a broad substrate specificity for anthracycline glycone substrates. Recombinant DoxA was purified to homogeneity from Streptomyces lividans transformed with a plasmid containing the Streptomyces sp. strain C5 doxA gene under the control of the strong SnpR-activated snpA promoter. The purified enzyme was a monomeric, soluble protein with an apparent Mr of 47,000. Purified DoxA catalyzed the 13-hydroxylation of 13-deoxydaunorubicin, the 13-oxidation of 13-dihydrocarminomycin and 13-dihydrodaunorubicin, and the 14-hydroxylation of daunorubicin. The pH optimum for heme activation was pH 7.5, and the temperature optimum was 30 degreesC. The kcat/Km values for the oxidation of anthracycline substrates by purified DoxA, incubated with appropriate electron-donating components, were as follows: for 13-deoxydaunorubicin, 22,000 M-1 x s-1; for 13-dihydrodaunorubicin, 14,000 M-1 x s-1; for 13-dihydrocarminomycin, 280 M-1 x s-1; and for daunorubicin, 130 M-1 x s-1. Our results indicate that the conversion of daunorubicin to doxorubicin by this enzyme is not a favored reaction and that the main anthracycline flux through the late steps of the daunorubicin biosynthetic pathway catalyzed by DoxA is likely directed through the 4-O-methyl series of anthracyclines.

Insulin-stimulated expression of c-fos, fra1 and c-jun accompanies the activation of the activator protein-1 (AP-1) transcriptional complex.

The activator protein-1 (AP-1) transcriptional complex is made up of members of the Fos (c-Fos, FosB, Fra1, Fra2) and Jun (c-Jun, JunB, JunD) families and is stimulated by insulin in several cell types. The mechanism by which insulin activates this complex is not well understood but it is dependent on the activation of the Erk1 and Erk2 isoforms of mitogen-activated protein kinases. In the current study we show that the AP-1 complex isolated from insulin-stimulated cells contained c-Fos, Fra1, c-Jun and JunB. The activation of the AP-1 complex by insulin was accompanied by (i) a transient increase in c-fos expression, and the transactivation of the ternary complex factors Elk1 and Sap1a, in an Erk1/Erk2-dependent fashion; (ii) a substantial increase in the expression of Fra1 protein and mRNA, which was preceded by a transient decrease in its electrophoretic mobility upon SDS/PAGE, indicative of phosphorylation; and (iii) a sustained increase in c-jun expression without increasing c-Jun phosphorylation on serines 63 and 73 or activation of the stress-activated kinase JNK/SAPK. In conclusion, insulin appears to stimulate the activity of the AP-1 complex primarily through a change in the abundance of the components of this complex, although there may be an additional role for Fra1 phosphorylation.

Central role for phosphatidylinositide 3-kinase in the repression of glucose-6-phosphatase gene transcription by insulin.

Transcription of the gene encoding the catalytic subunit of glucose-6-phosphatase (G6Pase) is stimulated by glucocorticoids and strongly repressed by insulin. We have explored the signaling pathways by which insulin mediates the repression of G6Pase transcription in H4IIE cells. Wortmannin, a phosphatidylinositide 3-kinase (PtdIns 3-kinase) inhibitor blocked the repression of G6Pase mRNA expression by insulin. However, both rapamycin, which inhibits p70S6 kinase activation, and PD98059, an inhibitor of mitogen-activated protein kinase activation, were without effect. Insulin inhibited dexamethasone-induced luciferase expression from a transiently transfected plasmid that places the luciferase gene under the control of the G6Pase promoter. This effect of insulin was mimicked by the overexpression of a constitutively active PtdIns 3-kinase but not by a constitutively active protein kinase B. Taken together, these data demonstrate that PtdIns 3-kinase activation is both necessary and at least partly sufficient for the repression of G6Pase expression by insulin, but neither mitogen-activated protein kinase nor p70S6 kinase are involved. In addition, activation of protein kinase B alone is not sufficient for repression of the G6Pase gene. These results imply the existence of a novel signaling pathway downstream of PtdIns 3 kinase that is involved in the regulation of G6Pase expression by insulin.

Evaluation of the EpiOcular((TM)) Tissue Model as an Alternative to the Draize Eye Irritation Test.

Cosmetic ingredients were tested to determine the ability of the EpiOcular(TM) tissue model to predict eye irritation potential. In vitro results were compared with historical Draize eye irritation records. Forty-three samples, consisting of 40 cosmetic raw ingredients of different type and physical form (i.e. liquids, powders, gels) were evaluated. Using the MTT cytotoxicity assay, an ET(50) value (effective time of exposure to reduce tissue viability to 50%) was determined for each sample. ET(50) values were categorized into four irritation groups: (a) non-irritating/minimal; (b) mild; (c) moderate; or (d) severe/extreme. Comparison of in vitro EpiOcular(TM) and in vivo Draize classifications showed that 63% (27 of 43 samples) were classified identically. Assay performance improved to 95% (41 of 43 samples) with the addition of samples overpredicted by a single irritation class. This evaluative exercise represents a conservative safety assessment. There were no underpredictions of eye irritation for any material in this study. Based on these results, use of the EpiOcular(TM) tissue model shows promise as an in vitro assay to assess the ocular irritation potential of cosmetic ingredients.