HPLC with electrochemical detection to examine the pharmacokinetics of baicalin and baicalein in rat plasma after oral administration of a Kampo medicine

A highly sensitive method for determining baicalin and baicalein in rat plasma was developed using micro HPLC with electrochemical detection (muHPLC-ED). Peak heights for baicalin and baicalein were found to be linearly related to the amounts injected, ranging from 2.2 pg to 4.5 ng (r = 0.997) and from 1.4 pg to 2.7 ng (r = 0.997), respectively. The detection limits (signal/noise ratio = 3) of the current method for baicalin and baicalein were 0.89 and 0.54 pg, respectively. The concentrations of baicalin, baicalein, and their conjugates in rat plasma after oral administration of 2.0 mg/kg of Saiko-keishi-to (TJ-10) were determined. The glucuronide and sulfate forms of baicalin and baicalein in plasma were hydrolyzed enzymatically using beta-glucuronidase and sulfatase, respectively, and the hydrolyzed solutions were extracted with a 0.1-mol/L phosphoric acid-ethyl acetate mixture (1:1, v/v). Based on the time courses of the concentrations of the free and conjugated forms of baicalin and baicalein in rat plasma after oral administration of Saiko-keishi-to, the pharmacokinetic parameters of C(max), t(max), and AUC(0-6 h) were obtained.     

Possible role of LRP1, a CCN2 receptor, in chondrocytes

Low density lipoprotein receptor (LDLR)-related protein 1 (LRP1/CD91) is one of the receptors of CCN2 that conducts endochondral ossification and cartilage repair. LRP1 is a well-known endocytic receptor, but its distribution among chondrocytes remains to be elucidated. We herein demonstrate for the first time that the distribution of LRP1 in chondrocytes except for hypertrophic chondrocytes in vivo and in vitro. Interestingly, the LRP1 levels were higher in mature chondrocytic HCS-2/8 and osteoblastic SaOS-2 than in other cells, whereas the other LDLR family members involved in ossification were detected at lower levels in HCS-2/8. It was interesting to note that in HCS-2/8, LRP1 was observed not only on the cell surface and in the cytoplasm, but also in the nucleus. Exogenously added CCN2 was incorporated into HCS-2/8, which was partially co-localized with LRP1, and targeted to the recycling endosomes and nucleus as well as the lysosomes. These findings suggest specific roles of LRP1 in cartilage biology.     

Mu-calpain is involved in the regulation of TNF-alpha-induced matrix metalloproteinase-3 release in a rheumatoid synovial cell line

Calpain is secreted by intra-articular synovial cells and degrades the main components of cartilage matrix proteins, proteoglycan, and collagen, causing cartilage destruction. Matrix metalloproteinase-3 (MMP-3) has also been detected in synovial fluid and serum, and is involved in the development and progression of rheumatoid arthritis by degradation of the extracellular matrix and cartilage destruction. To investigate the relationship between calpain and MMP-3 in rheumatic inflammation, we utilized the rheumatic synovial cell line, MH7A. Tumor necrosis factor (TNF-alpha) stimulation-induced increased expression of mu-calpain, m-calpain, and MMP-3 in these cells, as well as the release of calpain and MMP-3 into the culture medium. The calpain inhibitors, ALLN (calpain inhibitor I) and calpeptin, did not affect the intracellular expression of MMP-3, but reduced the secretion of MMP-3 in a concentration-dependent manner. Down-regulation of mu- but not m-calpain by small interfering RNAs abolished TNF-alpha-induced MMP-3 release from the synovial cells. These findings suggest that calpain, particularly mu-calpain, regulates MMP-3 release by rheumatic synovial cells, in addition to exerting its own degradative action on cartilage.     

Tetracycline residues and tetracycline resistance genes in groundwater impacted by swine production facilities

Antibiotics are used at therapeutic levels to treat disease; at slightly lower levels as prophylactics; and at low, subtherapeutic levels for growth promotion and improvement of feed efficiency. Over 88% of swine producers in the United States gave antimicrobials to grower/finisher pigs in feed as a growth promoter in 2000. It is estimated that ca. 75% of antibiotics are not absorbed by animals and are excreted in urine and feces. The extensive use of antibiotics in swine production has resulted in antibiotic resistance in many intestinal bacteria, which are also excreted in swine feces, resulting in dissemination of resistance genes into the environment. To assess the impact of manure management on groundwater quality, groundwater samples have been collected near two swine confinement facilities that use lagoons for manure storage and treatment. Several key contaminant indicators - including inorganic ions, antibiotics, and antibiotic resistance genes - were analyzed in groundwater collected from the monitoring wells. Chloride, ammonium, potassium, and sodium were predominant inorganic constituents in the manure samples and served as indicators of groundwater contamination. Based on these analyses, shallow groundwater has been impacted by lagoon seepage at both sites. Liquid chromatography-mass spectroscopy (LC-MS) was used to measure the dissolved concentrations of tetracycline, chlortetracycline, and oxytetracycline in groundwater and manure. Although tetracyclines were regularly used at both facilities, they were infrequently detected in manure samples and then at relatively trace concentrations. Concentrations of all tetracyclines and their breakdown products in the groundwater sampled were generally less than 0.5 microg/L. Bacterial tetracycline resistance genes served as distinct genotypic markers to indicate the dissemination and mobility of antibiotic resistance genes that originated from the lagoons. Applying PCR to genomic DNA extracted from the lagoon and groundwater samples, four commonly occurring tetracycline (tet) resistance genes - tet(M), tet(O), tet(Q), and tet(W) - were detected. The detection frequency of tet genes was much higher in wells located closer to and down-gradient from the lagoons than in wells more distant from the lagoons. These results suggested that in the groundwater underlying both facilities tetracycline resistance genes exist and are somewhat persistent, but that the distribution and potentially the flux for each tet gene varied throughout the study period.     

A fluorescent variant of a protein from the stony coral Montipora facilitates dual-color single-laser fluorescence cross-correlation spectroscopy

Dual-color fluorescence cross-correlation spectroscopy (FCCS) is a promising technique for quantifying protein-protein interactions. In this technique, two different fluorescent labels are excited and detected simultaneously within a common measurement volume. Difficulties in aligning two laser lines and emission crossover between the two fluorophores, however, make this technique complex. To overcome these limitations, we developed a fluorescent protein with a large Stokes shift. This protein, named Keima, absorbs and emits light maximally at 440 nm and 620 nm, respectively. Combining a monomeric version of Keima with cyan fluorescent protein allowed dual-color FCCS with a single 458-nm laser line and complete separation of the fluorescent protein emissions. This FCCS approach enabled sensitive detection of proteolysis by caspase-3 and the association of calmodulin with calmodulin-dependent enzymes. In addition, Keima and a spectral variant that emits maximally at 570 nm might facilitate simultaneous multicolor imaging with single-wavelength excitation.     

Epidemiological study of the relationship between sleep disturbances and somatic and psychological complaints among the Japanese general population

Sleep and Biological Rhythms - There have been only a few studies on the relationship between sleep disturbances and somatic and psychological complaints in Japanese people. In this study, we...     

Application of microchip electrophoresis in the analysis of RNA aptamer-protein interactions

DNA and RNA can be separated by microchip electrophoresis (ME) and detected using an intercalating fluorescent dye. The advantages of this method are short sensing times (<3 min), avoidance of a radioisotope labeling detection system, relatively low costs, and reduced labor intensity. In the present study, RNA aptamer-protein or -peptide interactions were analyzed using ME and the regression of free aptamers corresponding to unbound RNA was detected as the target protein or peptide increased in a dose-dependent manner. Our results demonstrate the applicability of this method to simple, rapid ligand screening in the interactions between oligonucleotides and their targets.     

Physiological properties of Cantor coding-like iterated function system in the hippocampal CA1 network

Cantor coding provides an information coding scheme for temporal sequences of events. In the hippocampal CA3–CA1 network, Cantor coding-like mechanism was observed in pyramidal neurons and the relationship between input pattern and recorded responses could be described as an iterated function system. However, detailed physiological properties of the system in CA1 remain unclear. Here, we performed a detailed analysis of the properties of the system related to the physiological basis of learning and memory. First, we investigated whether the system could be simply based on a series of on–off responses of excitatory postsynaptic potential (EPSP) amplitudes. We applied a series of three spatially distinct input patterns with similar EPSP peak amplitudes. The membrane responses showed significant differences in spatial clustering properties related to the iterated function system. These results suggest that existence of some factors, which do not simply depend on a series of on–off responses but on spatial patterns in the system. Second, to confirm whether the system is dependent on the interval of sequential input, we applied spatiotemporal sequential inputs at several intervals. The optimal interval was 30 ms, similar to the physiological input from CA3 to CA1. Third, we analyzed the inhibitory network dependency of the system. After GABA_A receptor blocker (gabazine) application, quality of code discrimination in the system was lower under subthreshold conditions and higher under suprathreshold conditions. These results suggest that the inhibitory network increase the difference between the responses under sub- and suprathreshold conditions. In summary, Cantor coding-like iterated function system appears to be suitable for information expression in relation to learning and memory in CA1 network.     

6-Carboxy-5,7-diarylcyclopenteno[1,2-b]pyridine derivatives: a novel class of endothelin receptor antagonists

Compounds (2-5) with a 6-carboxy-5,7-diarylcyclopentenopyridine skeleton were designed, synthesized, and identified as a new class of potent non-peptide endothelin receptor antagonists. The regio-isomer 2 was found to show potent inhibitory activity with an IC(50) value of 2.4 nM against (125)I-labeled ET-1 binding to human ET(A) receptors and a 170-fold selectivity for ET(A) over ET(B) receptors. Furthermore, 2 displayed more potent in vivo activity than did the indan-type compound 1 in a mouse ET-1 induced lethality model, suggesting the potential of 2 as a new lead structure. Derivatization on substituted phenyl groups at the 5- and 7-positions of 2 revealed that a 3,4-methylenedioxyphenyl group at the 5-position and a 4-methoxyphenyl group at the 7-position were optimal for binding affinity. Further derivatization of 2 by incorporating a substituent into the 2-position of the 4-methoxyphenyl group led to the identification of a more potent ET(A) selective antagonist 2p with an IC(50) value of 0.87 nM for ET(A) receptors and a 470-fold selectivity. In addition, 2p showed highly potent in vivo efficacy (AD(50): 0.04 mg/kg) in the lethality model.     

Synthesis and antibacterial evaluation of novel 2-[N-Imidoylpyrrolidinyl] carbapenems

The synthesis, antibacterial activity and DHP-susceptibility of a series of novel carbapenems, directly linked with heterocyclic moiety are described. Especially, the compounds linked pyrrolidine-carbapenem exhibited to have a good antibacterial activity against Staphylococcus aureus (MRSA) as well as Pseudomonas aeruginosa to maintain a good stability towards DHP-I.