Feast/famine regulatory proteins (FFRPs) comprise a single group of transcription factors systematically distributed throughout archaea and eubacteria. In the eubacterial domain in Escherichia coli, autotrophic pathways are activated and heterotrophic pathways are repressed by an FFRP, the leucine-responsive regulatory protein (Lrp), in some cases in interaction with other transcription factors. By sensing the concentration of leucine, Lrp changes its association state between hexadecamers and octamers to adapt the autotrophic or heterotrophic mode. The lrp gene is regulated so that the concentration of Lrp decreases in the presence of rich nutrition. In the archaeal domain a large part of the metabolism of Pyrococcus OT3 is regulated by another FFRP, FL11. In the presence of rich nutrition, the metabolism is released from repression by FL11; transcription of fl11 is terminated by FL11 forming octamers in interaction with lysine. When the nutrient is depleted, the metabolism is arrested by a high concentration of FL11; FL11 disassembles to dimers in the absence of lysine, and repression of transcription of fl11 is relaxed. Common characteristics of the master regulations by FL11 and Lrp hint at the prototype regulation once achieved in the common ancestor of all extant organisms. Mechanisms of discrimination by FFRPs between DNA sequences and also between co-regulatory molecules, mostly amino acids, and variations of transcription regulations observed with archaea and eubacteria are reviewed.
The analysis of nucleosome positions and transcription factor binding in chromatin is a central issue for understanding the mechanisms of gene expression in eukaryotes. Here, we have developed a footprinting technique, using multi-cycle primer extension with an infrared-fluorescence DNA sequencer, to analyze chromatin structure in isolated yeast nuclei and transcriptional activator binding in living yeast cells. Using this technique, the binding of the yeast activators Hap1 and Hap2/3/4/5 to their cognate sites was detectable as hypersensitive sites by in vivo UV-photofootprinting, and the locations of nucleosomes in yeast minichromosomes were determined by micrococcal nuclease mapping. We also applied this method to determine the position of the nucleosome in the 5S DNA fragment reconstituted in vitro. This technique allowed us to eliminate the use of radioactive materials and to perform experiments on common benches. Thus, the footprinting procedure established in this study will be useful to researchers studying DNA–protein interactions and chromatin structure in vivo and in vitro.
A novel phosphorylation motif for casein kinase 1 (CK1) in response to two sulfated lipids [sulfatide and cholesterol-3-sulfate (SCS)] was determined, using three functional proteins [myelin basic protein (MBP), tau protein (TP) and RhoA (a small GTPase)] and five synthetic MBP peptides as phosphate acceptors for the kinase in vitro. It was found that (i) MBP, p8 (positions 38—118) cleaved from MBP, and a synthetic peptide M103 were effectively phosphorylated by CK1δ in the presence of SCS; (ii) sulfatide in comparison with CH-3S highly enhanced autophosphorylation of CK1δ; (iii) SCS had a high binding affinity with MBP and peptide M103, but not other MBP peptides lacking K-G-R; and (iv) a novel consensus phosphorylation motif (K/R-X-K/R-X-X-S/T) for CK1 was identified among several SCS-binding proteins (SCS-BPs) and three CK1 isoforms (δ, ε and γ). The binding of SCS to two basic brain proteins (MBP and TP) resulted in the high stimulation of their phosphorylation by three CK1 isoforms (α, δ and ε), but not CK1γ. In contrast, an acidic protein (RhoA) was effectively phosphorylated by CK1δ in the presence of SCS, and also highly phosphorylated by CK1γ in the presence of sulfatide. Our results presented here suggest that (i) sulfatide may function as an effective stimulator for autophosphorylation of CK1; and (ii) cellular SCS-binding proteins, containing novel phosphorylation motifs for CK1, may be preferentially phosphorylated by CK1 with isoform specificity at the highly accumulated level of SCS in the brain.
The aim of this study was to characterize the gene expression profiles of mouse testis at different stages by performing large-scale complementary DNA (cDNA) analysis using DNA microarrays. The transcription profile of mouse testis was determined by comparing testis samples of mice aged 4, 9, 18, 35, 54 d and 6 months using Affymetrix Genechip mouse microarrays (430 v.2). Of the six comparative pairs of two neighboring spots examined, lots of differently expressed genes existed on day 18 versus day 9 and on day 35 versus day 18, indicating these genes may be involved in unique events during those periods. In mining the Genechip data, the gene expression profiles of 2058 genes represented in a gradually increased manner in the developmental stages of mice testis, and its validity was confirmed by the data of 21 known spermatogenesis-related testis-specific genes in our chip and the results of 10 random sample from 2058 genes produced by the sub-quantitative RT-PCR. So, we called these 2058 genes spermatogenesis-related genes. By informatics analysis, the gene expression clustering, gene ontology, KEGG pathway and domain regions were predicted for describing the potential roles that may play during mouse spermatogenesis. This study provides a molecular basis for the identity of spermatogenesis-related gene in mouse testis and leads to the elucidation of the molecular events underlying mammalian male reproduction.
The integrity of the genome is threatened by DNA damaging events such as radiation, viral infection and chemicals. Ionizing irradiation is known to cause genotoxic damage through the generation of reactive oxygen species (ROS) and nitrogen species (RNS) and we have found that a signaling pathway for the nuclear translocation of Translin is initiated in association and efficiently blocked by a specific inhibitor of nitric oxide synthase (NOS). This suggests the involvement of inducible nitric oxide synthase (iNOS)-derived nitric oxide (NO) in the nuclear translocation of Translin. To address the functional significance of Translin in the hematopoietic generation system after ionizing irradiation, we generated Translin-deficient (Translin−/−) mice and examined hematopoietic colony formation after sublethal ionizing irradiation. We thereby confirmed a severe delay of colony formation in the spleens of Translin−/− as compared with Translin+/+ mice. Taken together, the results suggest that Translin contributes to hematopoietic regeneration by acting as a sensor protein for radiation-induced damage.
The antimicrobial peptide LL-37 is generated from skin keratinocytes during infection of Gram-negative bacteria and exerts a microbicidal effect. LL-37 also causes functional changes in mast cells. Mast cells in the skin are involved in the innate immune system response against microbial infections via Toll-like receptors (TLRs), such as TLR4, which that is known to recognize lipopolysaccharide (LPS), a bacterial component. Thus, in the present study, we examined the effects of LL-37 on the expression of TLRs and the generation of cytokines on mast cells, and considered functional changes in the host defense system against bacteria. We observed that LL-37 increased the level of TLR4 mRNA and TLR4 protein, and that LL-37 induced the release of IL-4, IL-5 and IL-1β from mast cells. Cross-interaction between LL-37-triggered TLR4 augmentation and LL-37-inducible cytokine generation was also examined. Although the up-regulation of LL-37-inducible Th2 cytokines was cancelled by LPS, the augmentation of pro-inflammatory cytokine production was still observed. These findings indicate that LL-37 co-existing with the bacterial component switches mast cell function and directs human mast cells toward innate immunity. In conclusion, LL-37 may be a candidate modifier of the host defense against bacterial entry by serving as an alarm for sentinels such as mast cells.
Histone deacetylase (HDAC) inhibitors repress interleukin-2 (IL-2) gene expression in T cells and possess immunosuppressive activity in vivo. In addition to its immunosuppressive activity, HDAC inhibitors block GATA binding protein-1 (GATA-1) gene expression in megakaryocytes and elicit thrombocytopenia. In this report we state that for a given immunosuppressive dose of HDAC inhibitor, the ratio of GATA-1 reporter gene activity relative to IL-2 reporter gene assay (G/I ratio of measured IC50) can be predictive of a HDAC inhibitor's thrombocytopenic effect. This study utilized nine HDAC inhibitors at a minimal effective dose in a rat heterotopic cardiac transplantation model and the resultant G/I ratios and platelet depletion rates were highly correlated (r=0.933). These results indicate that calculation of G/I ratio can be a novel method for selecting immunosuppressive HDAC inhibitor having minimal thrombocytopenic effect which will benefit the search for new immunosuppressants of greater safety and efficacy.
In vitro effects of macrolide clarithromycin (CAM) on influenza A virus-infected cells were examined using plaque reduction assay by treating cells either before or after viral adsorption. The significant inhibitory effect on influenza virus infection was detected only when the cells were treated with CAM after viral adsorption. The predominant inhibitory effect was observed during 4—7th hour after viral adsorption using viral production assay. CAM did not exhibit inhibitory effects on influenza virus hemagglutination, membrane fusion and viral sialidase activities. These findings indicate that CAM acts on a middle to late stage of the viral replication cycle resulting in inhibition of progeny virus production from the infected cells.
AZ10992 is a novel paclitaxel–carboxymethyl (CM) dextran conjugate via a Gly–Gly–Phe–Gly linker with the molecular weight (MW) of 150 kDa. Our previous studies demonstrated that AZ10992 exerts strong antitumor activity against the human tumor xenografts that are highly refractory to paclitaxel, attributable to passive tumor targeting of released paclitaxel. This study examines the effects of carrier MW, anionic charge and drug-contents on the antitumor effects of AZ10992. To study antitumor effects, colon26 carcinoma-bearing BALB/c female mice received repeated (3 injections administered with 7 d intervals) intravenous administration of non-polymer-bound paclitaxel or paclitaxel–CM dextran conjugates. The results indicated that the conjugate comprising dextran T-110 (MW 110 kDa) with the degree of substitution (DS) value for the CM group of 0.50—0.55 per glucose residue and the drug contents of 5.5—6.5% (w/w) would be appropriate for AZ10992 regarding antitumor activity. Maximal tolerated dose (MTD) of AZ10992 was more than twice of non-polymer-bound paclitaxel. Furthermore, normal BALB/c female mice were treated with repeated (3 injections administered with 2 d intervals) intravenous administration of non-polymer-bound paclitaxel or AZ10992 at 50 mg/kg/d (based on the amount of paclitaxel to CM dextran) to study neurotoxicity. AZ10992 did not induce degeneration of myelin or swelling of Schwann cells in sciatic nerves.
The ATP-binding cassette (ABC) transporter protein subfamily B1 line (ABCB1) transporter P-glycoprotein (P-gp) plays an important role in the blood–brain barrier limiting a broad spectrum of substrates from entering the central nervous system. In the present study, the transport activity of P-gp for sertraline, desmethylsertralin, bupropion, and the major metabolites of bupropion, threo-amino alcohol (TB), erythro-amino alchhol (EB), and hydroxy metabolite (HB) was studied using an ATPase assay in expressed human P-gp membranes by measuring concentrations of inorganic Pi in expressed human P-gp membranes. Verapamil was included as a positive control. The Michaelis–Menten equation was used for characterizing the kinetic data. Sertraline and desmethylsertraline showed high affinity for P-gp. The Vmax/Km values of sertraline (1.6 min−1×10−3) and desmethylsertraline (1.4 min−1×10−3) were comparable with that of verapamil (1.7 min−1×10−3). Bupropion and its three metabolites showed very weak affinity for P-gp, with Vmax/Km values lower than 0.01 min−1×10−3. The results of the present study indicate that sertraline and desmethylsertraline have high affinity for P-gp, whereas bupropion and its three major metabolites TB, EB, and HB have very weak affinity for P-gp. These findings may help to explain observed drug–drug interactions among antidepressants.
This study describes the antinociceptive activity of extracts (methanolic (ME) and acetonic (AE)) and two phenolic compounds, 3,4,3′-trimethoxyflavellagic acid (1) and 3,4,3′-trimethoxy flavellagic acid 4′-O-glucoside (2), from Plinia glomerata leaves, against different experimental models of pain in mice. When evaluated against writhing test, by i.p. route, ME and AE presented calculated ID50 values (and respective confidence interval) of 3.28 (1.63—6.61) and 24.79 (16.57—37.09) mg/kg, respectively. Given by the oral route at 500 mg/kg, AE and ME extracts inhibited the abdominal constrictions by 60.5% and 35.3%, respectively. In the formalin test (10 mg/kg, i.p.), AE inhibited both phases of pain (45.6% in the first phase; 99.8% in the second phase) whereas ME inhibited 47.8% the first phase, and 92.6% the second phase. In the capsaicin test both extracts showed activity, with calculated ID50 values of 6.56 (5.69—7.56) and 7.68 (4.94—11.93) mg/kg for AE and ME, respectively. When evaluated against the hot-plate test, both extracts demonstrated activity, but only in high doses. Compound 2, when evaluated against the formalin test (10 mg/kg, i.p.), inhibited both phases of pain (77.6%, first phase; 62%, second phase) whereas 1 inhibited only the first phase, with inhibition of 70%. When tested in the capsaicin and glutamate tests, at 10 mg/kg, i.p., 1 and 2 caused inhibitions of 41.5% and 37.9%, and 37.7% and 54.5%, respectively. These results confirm previous studies carried out by our research group regarding the antinociceptive properties of P. glomerata, stimulating other studies on mechanism of action as well as the determination of additional active principles in this plant.
Hypericum perforatum extract (St. John's wort, SJW), Harpagophytum procumbens extract (HPE) and Grape seed proanthocyanidin extract (GSPE) have a broad spectrum of biological activities including antidepressant, anti-inflammatory or anti-oxidant effects. The aim of this study was to clarify antinociceptive properties of SJW, HPE and GSPE in mice with mechanisms that might potentially underlie these activities. Also, the effects of these herbal extracts on the antinociception and plasma and brain concentrations of morphine were examined. Oral pretreatment with SJW (100—1000 mg/kg) and HPE (30—300 mg/kg) attenuated significantly times of licking/biting both first and second phases of formalin injection in mice in the dose-dependent manner, and GSPE (10—300 mg/kg) suppressed second phase. Naloxone (5 mg/kg, s.c.) significantly attenuated antinociceptive effect of HPE but not SJW and GSPE. Formalin injection resulted in significant increase in the content of nitrites/nitrates (NOx) in mouse spinal cord. The rise of spinal NOx content by formalin was significantly attenuated by HPE and SJW. The pretreatment with SJW significantly potentiated an antinociceptive effect of morphine (0.3 mg/kg, s.c.), although concentrations of morphine in plasma and brain were not significantly changed by these herbal extracts. In conclusion, the present study has shown that SJW, HPE and GSPE exert significant antinociceptive effects in the formalin test of mice. In addition, opioidergic system seems to be involved in the antinociceptive effect of HPE but not SJW and GSPE. Furthermore, SJW potentiates morphine-induced antinociception possibly by pharmacodynamic interaction.
We examined the effect of chronic administration of imipramine and bupropion, monoamine reuptake inhibitors, on the duration of immobility in the forced swim test and serotonin (5-HT)2A receptor function in the form of 5-HT2A receptor mRNA levels in rats chronically treated with adrenocorticotropic hormone (ACTH). The immobility-decreasing effect of bupropion without imipramine did not influence the chronic ACTH treatment. The effect on the expression of 5-HT2A receptor mRNA of chronic ACTH treatment was decreased by bupropion, but not imipramine. These results suggest that bupropion has the effect of reducing immobility time in the forced swim test in the tricyclic antidepressant-resistant depressive model induced by chronic ACTH treatment in rats, and that decreased 5-HT2A receptor mRNA levels may be involved in this phenomenon.
Vitamin B12 contains a cobalt complex and accumulates at high levels in the liver. Vitamin B12 was examined for its hepatoprotective effect on dimethylnitrosamine-induced liver injury in mice. Vitamin B12 decreased the blood levels of aspartate aminotransferase and alanine aminotransferase, and clearly inhibited the overaccumulation of collagen fibrils. Reverse transcription-polymerase chain reaction (RT-PCR) analysis of the liver showed that the gene expression of α-smooth muscle actin and heat-shock protein 47, which are markers of fibrosis, were suppressed by vitamin B12 administration. Our findings indicate that vitamin B12 could be an effective hepatoprotective agent.
In this study, we investigated the effects of an Eriobotrya japonica seed extract (ESE) on mucositis using a 5-fluorouracil (5-FU)-induced mucositis hamster model. This model was prepared by intraperitoneally administering 90 mg/kg of 5-FU to hamsters on Day 1, scratching 1 cm2 of the left cheek pouch of hamsters with a wire brush on Days 2, 3, and 4, and intraperitoneally administering 60 mg/kg of 5-FU on Day 5. Mucositis was evaluated based on the mucositis score at the mucositis site, left cheek pouch thickness, histological findings on HE staining, and plasma lipid peroxide levels. On Day 10, the mucositis score and left cheek pouch thickness in the ESE group were significantly lower than those in the tap water group. Histologically, the two groups showed a defect of the cheek pouch epithelium on Day 6. On Day 10, epithelial injury and bacterial infection were noted in the tap water group. However, in the ESE group, similar findings were not observed. On Day 6, the plasma lipid peroxide level in the tap water group was significantly higher than that in the normal group. In the ESE group, the plasma lipid peroxide level was significantly lower than that in the tap water group. These results suggest that ESE is useful for treating chemotherapy-induced mucositis.
This study examined the comparative anticancer effects of flavonoids and diazepam in the cultured cancer cells. In the SNU-C4 colorectal and MDA-MB-231 breast adenocarcinoma cells, apigenin and fisetin, flavonoids, and diazepam inhibited cancer cell survival concentration and incubation-time dependently. Diazepam consistently inhibited FAS activity, a known anticancer mechanism of flavonoids, in a concentration dependent manner. Unlike diazepam, in highly aggressive breast MDA-MB-231 cells known to have a nuclear/perinuclear located PBR, PK11195, a specific PBR ligand enhanced the proliferation of cells, and the proliferative effect of PK11195 was reversed by an addition of lovastatin, a HMG-CoA reductase inhibitor. Diazepam- and flavonoids-induced cytotoxic activity in both cancer cell lines was not reduced by the addition of 5-fluorouracil (5-FU), a chemotherapeutic agent. Like flavonoids, diazepam inhibited the release of vascular endothelial growth factor (VEGF) and granulocyte-macrophage-colony stimulating factor (GM-CSF) into supernatants of cultured in the SNU-C4 and MDA-MB-231 cells. In conclusion, this study provided in vitro information on the safe use of sedative in oncologic patients.
A cladistic analysis of the medicinal plant Taxus, using the sequences of one chloroplast (trnS–trnQ spacer) and three nuclear taxadiene synthase (TS), 10-deacetylbaccatin III-10β-O-acetyltransferase (DBAT), and 18S rDNA) molecular markers, was carried out by distance, parsimony, likelihood, and Bayesian methods. Three of the four New World species (T. brevifolia, T. floridana and T. globosa) form a well-supported clade, whereas T. canadensis initially branches—appearing distantly related to both Old World taxa and New World species. In Asia, Taxus chinensis, T. mairei, T. sumatrana and T. wallichiana cluster together and are sister to a clade containing T. baccata and T. contorta. Taxus yunnanensis is more closely related to T. wallichiana than to four other Taxus species in our study from China; T. contorta is closer to the Euro-Mediterranean T. baccata than to the Asian species. This study provides a genetic method for authentication of economically important Taxus species and proposes a robust phylogenetic hypothesis for the genus. Using trnS–trnQ spacer sequences, we were able to distinguish T. mairei from all other species of Taxus.
In present paper, the properties of molecular authentication combined with the fingerprints of high performance liquid chromatography (HPLC) were validated by analyzing ten batches of Fructus Evodiae samples (the dried nearly ripe fruit of Evodia rutaecarpa (JUSS.) BENTH., Evodia rutaecarpa (JUSS.) BENTH. var. officinalis (DODE) HUANG or Evodia rutaecarpa (JUSS.) BENTH. var. bodinieri (DODE) HUANG). The results of this investigation show that the similarities of internal transcribed spacer (ITS) sequences were almost 100% in Evodia rutaecarpa (JUSS.) BENTH. var. bodinieri (DODE) HUANG, 97% in Evodia rutaecarpa (JUSS.) BENTH., and 96% in Evodia rutaecarpa (JUSS.) BENTH. var. officinalis (DODE) HUANG. The percentage of identity between the two groups of Evodia rutaecarpa (JUSS.) BENTH. var. bodinieri (DODE) HUANG and Evodia rutaecarpa (JUSS.) BENTH. var. officinalis (DODE) HUANG is almost 96%, but the identity among the group of these three species is only 73%. The results show that Fructus Evodiae comes from three species respectively. The fingerprints of HPLC show that Fructus Evodiae revealed 20 major common peaks. And the three species have almost the same chemical constituents. ITS sequence fingerprint combining the fingerprint of HPLC can not only be developed to identify and distinguish the three species in detail, but also can be used for optimizing location where Fructus Evodiae has much higher bioactive constituents and yield.
Saccharomyces boulardii is a nonpathogenic yeast with proven health benefits, some of them depending on its viability. However, the living yeast is sensitive to environmental conditions and its viability is less than 1% in the faeces after oral administration. Therefore, we assessed the survival conditions of S. boulardii in aqueous suspension and in its freeze-dried form and we formulated microspheres with the former and tablets with the latter in order to preserve the viability of the probiotic. While the viability of the yeast in aqueous suspension could be maintained for one year at −20 °C and +5 °C, increasing the temperature led to almost total mortality within 14 d at +40 °C and 4 d at +60 °C. The viability of the freeze-dried yeast was preserved for one year at +25 °C without moisture. With 75% relative humidity, the mortality was significant at 28 d at +25 °C and almost total within 1 d at +60 °C. In vitro, whereas less than 1% of non-encapsulated or non-tabletted S. boulardii survived after 120 min at pH 1.1, both formulations in microspheres and direct compression enabled to protect the yeast from degradation in HCl and to release it viable at pH 6.8. However, despite a similar release profile from both dosage forms, the compression led to a significant decrease in the viability of the freeze-dried yeast. In conclusion, although both formulations are efficient in protecting S. boulardii in acidic condition, microspheres provide a higher entrapment efficiency and a faster release of the viable probiotic in intestinal condition than matrix tablets.
Cisplatin is one of the most effective antineoplastic drugs, but it has undesirable side effects such as hepatotoxicity at high doses. This study investigated the protective effect of macelignan, isolated from Myristica fragrans HOUTT. (nutmeg), against cisplatin-induced hepatotoxicity and the possible mechanisms involved in these effects in mice. Pretreatment with macelignan for 4 d significantly prevented the increased serum enzymatic activities of alanine and aspartate aminotransferase in a dose-dependent manner. The results also showed that the protective effects of macelignan on cisplatin-induced hepatotoxicity may be associated with the mitogen activated protein kinase (MAPK) signaling pathway. Cisplatin-induced phosphorylation of c-Jun N-terminal kinase1/2 (JNK1/2) and extracellular signal-regulated kinase1/2 (ERK1/2) was abrogated by pretreatment with macelignan, however, that of p38 was not significantly affected. It was also found that macelignan attenuated the expression of phosphorylated c-Jun in cisplatin-treated mice. Accordingly, it is suggested that the hepatoprotective effects of macelignan could be related to activation of the MAPK signaling pathway, especially JNK and c-Jun, its substrate. The present findings suggest that co-treatment of cisplatin with macelignan may provide more advantage than cisplatin treatment alone in cancer therapy.
In order to estimate predisposing activity of oral application of beclomethasone dipropionate (BDP)-containing mucoadhesive films for oral candidiasis, the effects of BDP on growth of Candida albicans were examined in vivo and in vitro. Murine neutrophils inhibited the mycelial growth of C. albicans in vitro, but this anti-Candida activity was clearly suppressed by the presence of 10−6 M of BDP. In vitro, a BDP-release test showed that the amount of BDP released from BDP-containing films into the fluid phase increased in a time- and concentration-dependent manner and reached about 10—15% of the total amount of BDP in the film within 30 min. When the BDP-containing film was attached to the tongues of mice orally infected with C. albicans, oral infection by C. albicans deteriorated, but not as severely as in mice systemically immunosuppressed with prednisolone. Based on these findings, we also discuss the problems associated with the clinical application of BDP-film as an anti-inflammatory tool.
Ultraviolet (UV)B irradiation induces the production of matrix metalloproteinases (MMPs) by activating cellular signaling transduction pathways, which are responsible for the degradation or synthesis inhibition of collagenous extracellular matrix in connective tissues, causing skin photoaging. Using the human skin fibroblast (HS68) cell line in the present study, we investigated the inhibitory effects of fucoidan on MMP-1 expression by various in vitro experiments and elucidated the pathways of inhibition. Pretreatment with fucoidan inhibited UVB-induced MMP-1 expression in a dose-dependent manner. Extracellular signal regulated kinase (ERK) activation was markedly inhibited by treatment with fucoidan, though JNK activation was very slightly affected by fucoidan. We also found that fucoidan pretreatment significantly reduced MMP-1 mRNA expression in comparison with UVB irradiation only. In conclusion, our results demonstrate that fucoidan can mainly inhibit UVB-induced MMP-1 expression by inhibiting the ERK pathways. Therefore, fucoidan might be used as a potential agent for the prevention and treatment of skin photoaging.
Recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) has been produced in several different cell types, and has different properties depending on the production process used. There has been no report of glycosylation ratio or of the role of glycans for plant cells-derived rhGM-CSF. We investigated the terminal sialylation of rhGM-CSF produced in rice cells (rrhGM-CSF) using lectins. Glycosylation ratios of rrhGM-CSF and yeast-derived rhGM-CSF (yrhGM-CSF) were evaluated by chemical deglycosylation. After intravenous administration to rats, the clearance rates of rrhGM-CSF and yrhGM-CSF as well as deglycosylated forms of them were evaluated. In vitro bioactivities of rrhGM-CSF and yrhGM-CSF were measured in dose- and time-dependent manners. Although rrhGM-CSF does not have terminal sialic acids, the glycosylation ratio was two-fold higher than that of yrhGM-CSF, and rrhGM-CSF sustained longer than yrhGM-CSF in the blood circulation. Moreover, yrhGM-CSF and rrhGM-CSF have almost same bioactivity via dose- and time-dependent manners. Deglycosylated rrhGM-CSF was cleared faster than native forms, while deglycosylated yrhGM-CSF and yrhGM-CSF were similarly cleared in blood circulation. These results suggest that the glycans on rrhGM-CSF are superior to the glycans on yrhGM-CSF in terms of in vivo stability.
The aim of the present study was to investigate if the severity of illness affected the pharmacokinetics of cefuroxime in 11 children diagnosed with multiple organ system failure. The patients were assigned to a severely ill group (group 1), a very severely ill group (group 2), or a control group (group 0). Blood samples were taken and cefuroxime concentrations were measured in plasma by HPLC after the first intravenous infusion of 100 mg of cefuroxime per kg of body weight. The pharmacokinetic profile of cefuroxime exhibited both one and two compartmental distribution. Statistically significant differences between the pharmacokinetic parameters of the severe (group 1) and the very severe patients (group 2) were found, and significant differences (p<0.05) in the pharmacokinetic parameters between groups 1 and 2 vs. the control group were observed for most of the parameters analyzed. However, there was no statistical difference in clearance between group 1 and the control group. The data indicate that the pharmacokinetic differences determined by severity of disease are useful for establishing an individualized regimen dosage in children with multiple organ system failure.
In the process of lipolysis, adipocytes are stimulated by catecholamines through β1, β2, and β3 adrenergic receptors (ARs). So far, β2 and β3 AR polymorphisms have been reported related to obesity. However, the relation of β1AR polymorphisms to obesity has not been evaluated. In the present study, we examined whether βAR polymorphisms are associated with obesity-related phenotype in type II diabetic patients. Polymorphisms of β1Ser49Gly, β1Arg389Gly, β2Arg16Gly, β2Gln27Glu and β3Trp64Arg were genotyped in 188 type II diabetic patients by PCR-RFLP. Among these polymorphisms, β1Ser49Gly was found to be associated with obesity. Subjects with β1Gly49 allele showed higher body mass index (BMI) than those with Ser49/Ser49 genotype (24.7±3.7 vs. 23.4±3.3 kg/m2; p=0.031). Subjects with β1Gly49 allele were more frequently overweight (BMI≥25 kg/m2) compared with β1Ser49 homozygous group (42.1 vs. 24.4%, p=0.015). By multiple linear regression analysis, β1Ser49Gly polymorphism was independently associated with higher BMI (p=0.019, β=0.166). Our data indicate that the Gly49 allele in β1AR is associated with higher BMI in type II diabetic patients. Genotyping for β1Ser49Gly polymorphism in type II diabetic patients may have clinical benefit to predict obesity, thereby contributing to the prevention of insulin resistance.
In 133 patients suspected of hypersensitivity to drugs and 102 control patients without hypersensitivity to drugs, the identification of allergenic drugs was performed by the drug-induced lymphocyte stimulation test (DLST) and the leukocyte migration test (LMT) to compare their usefulness in identifying drug allergies. In the 133 subject patients, the positive rate was 24.8% on the DLST and 60.9% on the LMT (agreement rate; 77.4%); thus, the LMT showed a significantly higher positive rate than the DLST (p<0.000001, χ2-test). In the 102 control patients, the positive rates on the DLST and LMT were 6.9%. In addition, the LMT showed a higher positive rate than the DLST for many hypersensitivity symptoms such as skin eruptions and hepatic injury, and for many drug efficacy categories of the suspected drugs such as antibacterial drugs, etc. Furthermore, the positive rate of the DLST did not change when adjusted for the patients' serum and sex, while that of the LMT increased when adjusted for the patients' serum and was found to be higher in females than in males. Our findings indicate that the LMT may be more useful than the DLST in identifying the causative drug in drug allergies and that its interpretation is influenced by the patient's serum and sex.
Pharmacokinetic profiles of probucol were evaluated after oral administration of the various nanosuspensions in rats. Probucol nanoparticles were prepared by co-grinding with various molecular weights of polyvinylpyrrolidone (PVP K12, PVP K17 and PVP K30) and sodium dodecyl sulfate (SDS). The average particle sizes of probucol after dispersing the ternary ground mixtures (GMs), probucol/PVP K12/SDS, probucol/PVP K17/SDS and probucol/PVP K30/SDS into water were 28, 75 and 89 nm respectively. The ternary GM suspensions with PVP K17/SDS and PVP K30/SDS were stable at 25 °C. However the particle size of probucol from the ternary GM with PVP K12/SDS gradually increased. Pharmacokinetic profiles of probucol indicated that variation in particle surface condition covered with PVP and SDS in addition to the particle size affected the improvement of in vivo absorption of probucol. The ternary GM with PVP K12/SDS exhibited a superior improvement of probucol absorption compared to the GMs with PVP K17/SDS and PVP K30/SDS. The binary GM with PVP or SDS and physical mixtures with PVP and/or SDS did not show significant differences in the area under the plasma concentration–time curve compared to the unprocessed probucol. In conclusion, preparation of probucol nanoparticles by co-grinding with PVP K12 and SDS could be a promising method for bioavailability enhancement.