Drug governs the morphology of polyalkylated block copolymer aggregates.

Polyalkylated copolymers based on mPEG-b-(AGE-C6,12 or 18)25 have been used to formulate clinically relevant concentrations of doxorubicin (DOX) and the impact of drug incorporation on copolymer aggregation behaviour was examined. The copolymer aggregates were analyzed by various microscopy techniques (TEM, cryo-TEM and AFM) and scattering methods (SANS, DLS). In the absence of the drug, the copolymers formed largely non-spherical aggregates (i.e. cylinders, vesicles). Drug incorporation during copolymer aggregate formation directed the formation of only spherical aggregates. As well, the nature of the core-forming block was found to influence drug release and cytotoxicity of the formulations.

Temporal variability of glucocorticoid receptor activity is functionally important for the therapeutic action of fluoxetine in the hippocampus.

Previous studies have shown inconsistent results regarding the actions of antidepressants on glucocorticoid receptor (GR) signalling. To resolve these inconsistencies, we used a lentiviral-based reporter system to directly monitor rat hippocampal GR activity during stress adaptation. Temporal GR activation was induced significantly by acute stress, as demonstrated by an increase in the intra-individual variability of the acute stress group compared with the variability of the non-stress group. However, the increased intra-individual variability was dampened by exposure to chronic stress, which was partly restored by fluoxetine treatment without affecting glucocorticoid secretion. Immobility in the forced-swim test was negatively correlated with the intra-individual variability, but was not correlated with the quantitative GR activity during fluoxetine therapy; this highlights the temporal variability in the neurobiological links between GR signalling and the therapeutic action of fluoxetine. Furthermore, we demonstrated sequential phosphorylation between GR (S224) and (S232) following fluoxetine treatment, showing a molecular basis for hormone-independent nuclear translocation and transcriptional enhancement. Collectively, these results suggest a neurobiological mechanism by which fluoxetine treatment confers resilience to the chronic stress-mediated attenuation of hypothalamic-pituitary-adrenal axis activity.

Family history of diabetes and the risk of subclinical atherosclerosis.

AIM: This study investigated the influence of a family history of diabetes on the risk of subclinical coronary atherosclerosis according to coronary computed tomography angiography (CCTA) in asymptomatic individuals. METHODS: A total of 6434 consecutive asymptomatic individuals with no prior history of coronary artery disease voluntarily underwent CCTA evaluation as part of a general health examination. Coronary atherosclerotic plaque and significant coronary artery stenosis (degree of stenosis ≥50%) on CCTA were assessed. Logistic regression analysis was used to determine the association between a family history of diabetes and atherosclerotic plaque or significant coronary artery stenosis according to the degree of diabetes (normal, prediabetic and diabetic). RESULTS: Mean age of study participants was 53.7±7.6 years, and 4694 (73.0%) were male. A total of 1593 (24.8%) participants had a family history of diabetes in a first-degree relative. Among the study participants, 1115 (17.3%), 3122 (48.5%) and 2197 (34.1%) were categorized as diabetic, prediabetic and normal, respectively. In diabetic participants, after stepwise adjustments for clinical and laboratory variables, a family history of diabetes was significantly associated with non-calcified plaque (P<0.05 for all), but did not appear to be associated with either calcified or mixed plaques or with significant coronary artery stenosis (P>0.05 for all). In prediabetic and normal participants, a family history of diabetes was not associated with either atherosclerotic plaque or significant coronary artery stenosis (P>0.05 for all). CONCLUSION: In asymptomatic diabetic individuals, a family history of diabetes is consistently associated with non-calcified coronary plaque after adjusting for risk factors.

Real-time in vivo bioluminescence imaging of lentiviral vector-mediated gene transfer in mouse testis.

Although much research has focused on transferring exogenous genes into living mouse testis to investigate specific gene functions in spermatogenic, Sertoli, and Leydig cells, relatively little is known regarding real-time gene expression in vivo. In this study, we constructed a bicistronic lentiviral vector (LV) encoding firefly luciferase and enhanced green fluorescence protein (EGFP); this was a highly efficient in vivo gene transfer tool. After microinjecting LV into the seminiferous tubules the ICR mouse testis, we detected luciferase and EGFP expression in vivo and ex vivo in the injected tubules using bioluminescence imaging (BLI) with the IVIS-200 system and fibered confocal fluorescence microscopy (CellViZio), respectively. In addition, with an in vivo BLI system, luciferase expression in the testis was detected for approximately 3 mo. Furthermore, EGFP expression in seminiferous tubules was confirmed in excised testes via three-dimensional fluorescent imaging with a confocal laser-scanning microscope. With immunostaining, EGFP expression was confirmed in several male germ cell types in the seminiferous tubules, as well as in Sertoli and Leydig cells. In conclusion, we demonstrated that real-time in vivo BLI analysis can be used to noninvasively (in vivo) monitor long-term luciferase expression in mouse testis, and we verified that EGFP expression is localized in seminiferous tubules after bicistronic LV-mediated gene transfer into mouse testes. Furthermore, we anticipate the future use of in vivo BLI technology for real-time study of specific genes involved in spermatogenesis.

Immunoreactivity and protein levels of olfactory marker protein and tyrosine hydroxylase are not changed in the dog main olfactory bulb during normal ageing.

The immunoreactivity and protein expression of olfactory marker protein (OMP) and tyrosine hydroxylase (TH) in the main olfactory bulb (MOB) of the dog during normal ageing was investigated. OMP immunolabelling was observed only in nerve bundles of the olfactory nerve (ONL) and glomerular layers (GL) and there was no OMP expression within cell bodies of any layer. TH immunolabelling was detected in all layers of the MOB except for the ONL. Most of the neurons expressing TH were distributed in the juxtaglomerular region and had a morphological appearance consistent with periglomerular, external tufted or superficial short axon cells. Dendrites of TH-immunoreactive neurons were closely apposed to OMP-immunoreactive nerve bundles within the glomeruli. There was no significant age-related loss of OMP and TH immunoreactivity and protein concentrations of these molecules were consistent in dogs of different ages. These results suggest that olfactory signal transduction to the GL via axons of olfactory receptor neurons remains unchanged during ageing in the dog.

Stress-induced changes in primate prefrontal profiles of gene expression.

Stressful experiences that consistently increase cortisol levels appear to alter the expression of hundreds of genes in prefrontal limbic brain regions. Here, we investigate this hypothesis in monkeys exposed to intermittent social stress-induced episodes of hypercortisolism or a no-stress control condition. Prefrontal profiles of gene expression compiled from Affymetrix microarray data for monkeys randomized to the no-stress condition were consistent with microarray results published for healthy humans. In monkeys exposed to intermittent social stress, more genes than expected by chance appeared to be differentially expressed in ventromedial prefrontal cortex compared to monkeys not exposed to adult social stress. Most of these stress responsive candidate genes were modestly downregulated, including ubiquitin conjugation enzymes and ligases involved in synaptic plasticity, cell cycle progression and nuclear receptor signaling. Social stress did not affect gene expression beyond that expected by chance in dorsolateral prefrontal cortex or prefrontal white matter. Thirty four of 48 comparisons chosen for verification by quantitative real-time polymerase chain reaction (qPCR) were consistent with the microarray-predicted result. Furthermore, qPCR and microarray data were highly correlated. These results provide new insights on the regulation of gene expression in a prefrontal corticolimbic region involved in the pathophysiology of stress and major depression. Comparisons between these data from monkeys and those for ventromedial prefrontal cortex in humans with a history of major depression may help to distinguish the molecular signature of stress from other confounding factors in human postmortem brain research.

A repeated batch process for cultivation of Bifidobacterium longum.

A repeated batch process was performed to culture Bifidobacterium longum CCRC 14634. An on-line device, oxidation-reduction potential (ORP), was used to monitor cell growth and uptake of nutrients in the culture. The ORP of the culture medium decreased substantially during fermentation until nutrients were depleted. Six cycles of batch fermentation using ORP as a control parameter were successfully carried out. As soon as ORP remained constant or increased, three-quarters of the broth was removed, and the same volume of fresh medium was fed to the fermenter for a new cycle of cultivation. Average cell concentrations of 1.9 x 10(9) and 3.4 x 10(9) cfu ml(-1) for repeated batch fermentation in MRS (Lactobacilli MRS broth) and WY (containing whey hydrolyzates, yeast extract, l-cysteine) medium, respectively, were achieved. Cell mass productivities for batch, fed-batch and repeated batch fermentation using MRS medium were 0.51, 0.41, and 0.64 g l(-1) h(-1), respectively, and those for batch and repeated batch using WY medium were 0.76, 0.99 g l(-1) h(-1), respectively. The results indicate a possible industrial process to culture Bifidobacteria sp.

Glucocorticoid responsiveness of the rat phenylethanolamine N-methyltransferase gene.

Two newly identified, overlapping (1 bp) glucocorticoid response elements (GREs) at -759 and -773 bp in the promoter of the rat phenylethanolamine N-methyltransferase (PNMT; EC 2.1.1.28) gene are primarily responsible for its glucocorticoid sensitivity, rather than the originally identified -533-bp GRE. A dose-dependent increase in PNMT promoter activity was observed in RS1 cells transfected with a wild-type PNMT promoter-luciferase reporter gene construct and treated with dexamethasone (maximum activation at 0.1 microM). The type II glucocorticoid receptor antagonist RU38486 (10 microM) fully inhibited dexamethasone (1 microM) activation of the PNMT promoter, consistent with classical glucocorticoid receptors mediating corticosteroid-stimulated transcriptional activity. Relative IC(50) values from gel mobility shift competition assays showed that the -759-bp GRE has a 2-fold greater affinity for the glucocorticoid receptor than the -773-bp GRE. Site-directed mutation of the -533-, -759-, and -773-bp GREs alone or in tandem demonstrated that the -759-bp GRE was also functionally more important, but both the -759- and -773-bp GREs are required for maximum glucocorticoid responses. Moreover, the -533-bp GRE, rather than increasing glucocorticoid sensitivity of the promoter, may limit corticosteroid responsiveness mediated via the -759- and -773-bp GREs. Finally, the glucocorticoid receptor bound to the -759- and -773-bp GREs interacts cooperatively with Egr-1 and/or AP-2 to stimulate PNMT promoter activity in RS1 cells treated with dexamethasone. In contrast, glucocorticoid receptors bound to the -533-bp GRE only seem to participate in synergistic activation of the PNMT promoter through interaction with activator protein 2.

Influence of serum-free culture conditions on steroid 5alpha-reductase mRNA expression in rat C6 glioma cells.

Immunocytochemical studies previously showed that serum deprivation resulted in the appearance of steroid 5alpha-reductase (5alpha-R) in the cytoplasm of rat C6 glioma cells. To determine whether this increase in cytoplasmic 5alpha-R was due to changes in 5alpha-R gene expression, the effect of serum deprivation on 5alpha-R mRNA expression was examined. No significant change in the mRNA levels was observed in cells grown in serum-free culture medium. Therefore, the appearance of 5alpha-R immunoreactivity in the cell cytoplasm observed under serum-free conditions is probably not due to changes in 5alpha-R gene expression.

Phenylethanolamine N-methyltransferase gene expression. Sp1 and MAZ potential for tissue-specific expression.

Phenylethanolamine N-methyltransferase (PNMT) promoter-luciferase reporter gene constructs (pGL3RP863, pGL3RP444, and pGL3RP392) transfected into COS1, RS1, PC12, NIH/3T3, or Neuro2A cells showed the highest basal luciferase activity in the Neuro2A cells. DNase I footprinting with Neuro2A cell nuclear extract identified protected PNMT promoter regions spanning the -168/-165 and -48/-45 base pair Sp1/Egr-1 binding sites. Gel mobility shift assays and transient transfection assays using site-directed mutant PNMT promoter-luciferase reporter gene constructs indicated that the elevated basal luciferase activity in the Neuro2A cells was mediated by Sp-1. Furthermore, activation of the PNMT promoter by Sp1 depends on both its binding affinity for its cognate target sequences and its intracellular concentrations. When Sp1 levels were increased through an expression plasmid, luciferase reporter gene expression rose well beyond basal wild-type levels, even with either Sp1 binding element mutated. Finally, another transcription factor expressed in the Neuro2A cells competes with Sp1 by interacting with DNA sequences 3' to the -48 base pair Sp1 site to prevent Sp1 binding and induction of the PNMT promoter. The DNA consensus sequence, Southwestern analysis, and gel mobility shift assays with antibodies identify MAZ as the competitive factor. These findings suggest that Sp1 may potentially contribute to the tissue-specific expression of the PNMT gene, with the competition between Sp1 and MAZ conferring additional tissue-specific control.

Partial characterization and cloning of leuconocin J, a bacteriocin produced by Leuconostoc sp. J2 isolated from the Korean fermented vegetable Kimchi.

Leuconostoc sp. J2, isolated from naturally fermented Kimchi, produced a bacteriocin which was named leuconocin J. This bacteriocin exhibited an inhibitory activity against several lactic acid bacteria and some food-borne pathogens. The antimicrobial substance was secreted into the medium during the late log phase. It appears to be proteinaceous since its activity was completely inactivated by a range of proteolytic enzymes, and it was also relatively heat-stable. The bacteriocin was partially purified by ammonium sulphate precipitation, following dialysis. The apparent molecular mass of partially purified bacteriocin, as indicated by activity detection after Tricine-SDS-PAGE, was 2.5-3.5 kDa. Leuconostoc sp. J2 plasmid DNA digested by EcoRI was cloned into pUC118 and transformed into Escherichia coli DH5 alpha. Phenotypic expression of the bacteriocin production was detected in transformants harboring pULBJ5.5. Finally, Southern blotting with the 2.3 kb insert as a probe against plasmid digests of Leuconostoc sp. J2 revealed that the cloned foreign DNA originated from Leuconostoc sp. J2.

Cloning and sequencing of beta-1,4-endoglucanase gene (celA) from Pseudomonas sp. YD-15.

A beta-1,4-endoglucanase gene (celA) from Pseudomonas sp. YD-15 was cloned in Escherichia coli DH5 alpha and its nucleotide sequence determined. The open reading frame of celA was 1830 base pairs and the enzyme was composed of 609 amino acids with a molecular weight of 63,617 Da. The deduced amino acid sequence and putative active site of CelA had high amino acid homology with family E cellulases. By dot blot analysis, the induction of celA according to carbon sources was determined. The transcripts hybridizing to the internal fragment of celA were detected in total RNA isolated from Pseudomonas sp. YD-15 cells grown on avicel and glycerol, but not from cells grown on glucose and cellobiose.

Phenylethanolamine N-methyltransferase gene expression: synergistic activation by Egr-1, AP-2 and the glucocorticoid receptor.

The gene encoding the epinephrine synthesizing enzyme, phenylethanolamine N-methyltransferase (PNMT), is transcriptionally activated by Egr-1, AP-2, and the glucocorticoid receptor (GR). Stimulation by AP-2 requires its synergistic interaction with an activated GR. The present studies show that the GR also cooperates with Egr-1 or the combination of Egr-1 and AP-2 to activate the PNMT promoter. Together Egr-1, AP-2, and the GR can induce PNMT promoter-mediated luciferase reporter gene expression beyond the sum of their independent contributions as well as synergistically activate the endogenous PNMT gene leading to marked increases in PNMT mRNA. Examination of the effects of mutation of the AP-2 or Egr-1 binding sites on PNMT promoter activation by DEX and the factor binding to the remaining intact site or by all three transcriptional activators showed changes in luciferase reporter gene expression which suggest that DNA structure may be altered thereby reducing or enhancing synergistic activation. It also appears that the -165 bp Egr-1 site may not be critical for the synergism observed between Egr-1, AP-2 and the GR. When the glucocorticoid response element (GRE) within the PNMT promoter was mutated, PNMT promoter activation by Egr-1 and DEX, AP-2 and DEX or all three showed both inhibition and enhancement, even when the GRE was completely eliminated. These observations indicate that induction of PNMT gene transcription may occur either through GR interaction with other transcriptional proteins after binding to its cognate GRE or through direct protein-protein interaction in the absence of GRE binding. While the mechanisms by which Egr-1 and the GR and Egr-1, AP-2 and the GR function cooperatively to stimulate PNMT promoter activity remain to be elucidated, this synergistic stimulation of the PNMT promoter by these factors may provide important in vivo and in vitro regulatory control of the PNMT gene.

Determination of medrogestone in plasma by high-performance liquid chromatography.

Interference with the UV absorbance of medrogestone by endogenous steroids in plasma was prevented by reacting plasma with oxalyl chloride. The reduction of interference was effective when oxalyl chloride was in the range 10-50 microl/ml plasma. Reaction of oxalyl chloride with plasma for 10 min could reduce interference approximately 5.5-fold, and there was no significant reduction after 30 min. The limit of quantitative concentration for medrogestone in HPLC was 1 ng/ml. The standard curves were linear with the correlation coefficient greater than 0.999 in the range of 1-30 ng/ml. The coefficients of variation of both intra- and inter-day mean values were <12% and <10% of the actual values, respectively. The developed method for plasma sample preparation and the evaluated HPLC condition were further applied to an in vivo pharmacokinetic study.

Glucocorticoid-dependent action of neural crest factor AP-2: stimulation of phenylethanolamine N-methyltransferase gene expression.

AP-2 is a vertebrate transcription factor expressed in neural crest cells and their derivative tissues, including the adrenal medulla, where epinephrine is produced. AP-2 is shown to stimulate expression of the gene encoding the epinephrine biosynthetic enzyme phenylethanolamine N-methyltransferase (PNMT). However, stimulation of the PNMT gene by AP-2 requires glucocorticoids and appears to be mediated through the interaction of AP-2 with activated type II glucocorticoid receptors. Mutation of AP-2 and/or glucocorticoid receptor binding elements within the PNMT promoter disrupts the ability of AP-2 and glucocorticoids to induce PNMT promoter activity. These findings suggest, in the case of PNMT, that AP-2 stimulates gene expression through a novel glucocorticoid-dependent mechanism.

Cloning and sequence analysis of a novel member of the single-stranded DNA binding protein family.

A single-stranded DNA binding protein (SSB) was isolated which has unusual amino acid residues at sites previously shown to be highly conserved and critical for DNA binding. Sequence analysis suggested that these residues are characteristic of a novel class of SSBs from Gram-positive bacteria.

A distal activation domain is critical in the regulation of the rat androgen receptor gene promoter.

The far upstream region of the rat androgen receptor (AR) gene has been cloned, and the nucleotide sequence up to -2656 bp established. Nested deletion mutants of rat AR 5' flanking sequences were ligated to the luciferase reporter gene, and their promoter activities were examined in transfected COS1 cells. Results show a critical cis-acting domain located between positions -960 and -940. Deletion of this cis element resulted in a greater than 90% decrease in the promoter activity. A nuclear protein that specifically binds to this 21-nucleotide sequence was identified by gel mobility shift analysis. The -960/-940 cis element has no identify to the binding sequence of any known transcription factor. Furthermore, the cognate binding protein is present in both rat and human (HeLa) cell nuclear extracts. We conclude that a novel trans-activator interacting at the -960/-940 region plays a critical role in the regulation of AR gene expression.

Estrogen sulfotransferase of the rat liver: complementary DNA cloning and age- and sex-specific regulation of messenger RNA.

Mammalian estrogen sulfotransferase (EST; EC 2.8.2.4) sulfurylates the hydroxyl group of estrogenic steroids by transferring the sulfate from a cosubstrate adenosine 3'-phosphate-5'-phosphosulfate. Sulfurylated steroids do not bind to the estrogen receptor with high affinity and, therefore, are hormonally inactive. We have purified rat liver EST and developed monoclonal antibody to this enzyme. By immunoscreening a lambda gt-11 expression library constructed from male rat liver cDNAs, the cDNA clone corresponding to EST was identified and isolated. A recombinant expression plasmid (pCMV5) containing this cDNA insert when transfected into COS-7 cells generated both immunologically and enzymatically active EST. With the help of this cDNA probe, we have explored the regulation of the EST mRNA in the liver and the possible role of this enzyme in sex hormone action. During the lifespan of male rats, only the young adult animals show hepatic androgen responsiveness. Also, estrogenic hormones strongly antagonize androgen action in the rat liver. Northern blot analysis of liver RNA derived from male rats of different ages shows that the androgen sensitivity of young adult animals is associated with a high expression of EST mRNA. During the same period, mRNA corresponding to dehydroepiandrosterone sulfotransferase is markedly (approximately 10-fold) down-regulated. Such a correlation is in concordance with the role of these enzymes in the maintenance of hepatic androgen sensitivity during young adult life by inactivating the estrogenic and sparing the androgenic steroids. Furthermore, the increase in the hepatic androgen sensitivity of androgen-treated female rats is also associated with the induction of EST.(ABSTRACT TRUNCATED AT 250 WORDS)