Simple and sensitive bacterial quantification by a flow-based kinetic exclusion fluorescence immunoassay

A flow-based immunoassay system utilizing secondary-antibody coated microbeads and Cy5-secondary antibody for signal production was successfully developed to quantitate target bacteria with a kinetic exclusion assay (KinExA 3000 Instrument). It directly measured the concentration of unliganded antibody separated from the equilibrated mixture of antibody and bacteria through a 0.2 microm polyethersulfone membrane, enabling it to quantify the concentration of bacteria. The novel method demonstrated the qualities of rapidness, sensitivity, high accuracy and reproducibility, and ease to perform. Detection of Pseudomonas aeruginosa and Staphylococcus aureus was accomplished with low detection limits of 4.10 x 10(6) and 5.20 x l0(4)cells/mL, respectively, with an assay time of less than 15 min. The working ranges for quantification were 4.10 x l0(6) to 1.64 x l0(10)cells/mL for P. aeruginosa, and 5.20 x l0(4) to 1.04 x l0(9)cells/mL for S. aureus. It yielded an assay with at least 10-fold greater sensitivity than ELISA and could correctly assess the concentration of predominant bacterium spiked in the mixture of P. aeruginosa and S. aureus. With this reliable platform, the average amount of antibody bound by one cell in the maximum capability could be further provided: (1.6-2.5) x l0(5) antibodies for one P. aeruginosa cell and (2.2-2.7) x l0(8) antibodies for one S. aureus cell. The KinExA system is flexible to determine different kinds of bacteria conveniently by using anti-mouse IgG as the same immobilizing agent. However, a higher specificity of the antibodies to the target bacteria will be required for the use of this system with higher detection sensitivity.     

Beneficial effect of GB virus C co-infection in Human Immunodeficiency Virus type 1-infected individuals

Several reports have documented a better prognosis for HIV‐1‐infected patients co‐infected with GBV‐C, while other reports have contradicted such findings with the result that this issue remains controversial. We attempted to clarify the complicated status of the effect of GBV‐C co‐infection on HIV‐1‐infected patients. GBV‐C RNA was detected in 37 samples in 182 HIV‐1‐infected patients (20.3%) using RT/nested PCR. Of these, 3 were determined to be GBV‐C genotype 1, 12 were genotype 2, and the remaining 22 were genotype 3. The GBV‐C viral load quantified by real‐time PCR ranged from 7.8 × 10³ to 3.3 × 10⁶ copies/ml. Weakly negative correlation was observed between GBV‐C viral load and HIV‐1 viral load in 19 HAART‐naïve patients, indicating that a higher GBV‐C viral load is associated with a greater suppression of HIV‐1 replication. A previously published in vitro study suggested that GBV‐C infection would induce up‐regulation of RANTES, leading to suppression of HIV‐1 replication. However, in our present study, the blood RANTES level was significantly lower in the GBV‐C co‐infected group than in the uninfected group (190–9,959 vs. 264–31,038 pg/ml, P=0.004). Our results suggested that a suppression of HIV‐1 replication by GBV‐C co‐infection is not mediated by up‐regulated RANTES, and thus call for another as yet unknown factor.     

Molecular analysis of RANKL-independent cell fusion of osteoclast-like cells induced by TNF-alpha, lipopolysaccharide, or peptidoglycan

Focusing on the final step of osteoclastogenesis, we studied cell fusion from tartrate-resistant acid phosphatase (TRAP)-positive mononuclear cells into multinuclear cells. TRAP-positive mononuclear cells before generation of multinuclear cells by cell fusion were differentiated from RAW264.7 cells by treatment with receptor activator of nuclear factor kappa B ligand (RANKL), and then the cells were treated with lipopolysaccharide (LPS), followed by culturing for further 12 h. LPS-induced cell fusion even in the absence of RANKL. Similarly, tumor necrosis factor (TNF)-alpha and peptidoglycan (PGN) induced cell fusion, but M-CSF did not. The cell fusion induced by RANKL, TNF-alpha, and LPS was specifically blocked by osteoprotegerin (OPG), anti-TNF-alpha antibody, and polymyxin B, respectively. LPS- and PGN-induced cell fusion was partly inhibited by anti-TNF-alpha antibody but not by OPG. When TRAP-positive mononuclear cells fused to yield multinuclear cells, phosphorylation of Akt, Src, extracellular signal-regulated kinase (ERK), p38MAPK (p38), and c-Jun NH2-terminal kinase (JNK) was observed. The specific chemical inhibitors LY294002 (PI3K), PP2 (Src), U0126 (MAPK-ERK kinase (MEK)/ERK), and SP600125 (JNK) effectively suppressed cell fusion, although SB203580 (p38) did not. mRNA of nuclear factor of activated T-cells c1 (NFATc1) and dendritic cell-specific transmembrane protein (DC-STAMP) during the cell fusion was quantified, however, there was no obvious difference among the TRAP-positive mononuclear cells treated with or without M-CSF, RANKL, TNF-alpha, LPS, or PGN. Collectively, RANKL, TNF-alpha, LPS, and PGN induced cell fusion of osteoclasts through their own receptors. Subsequent activation of signaling pathways involving PI3K, Src, ERK, and JNK molecules was required for the cell fusion. Although DC-STAMP is considered to be a requisite for cell fusion of osteoclasts, cell fusion-inducing factors other than DC-STAMP might be necessary for the cell fusion.     

Gene conversion between mammalian CCR2 and CCR5 chemokine receptor genes: a potential mechanism for receptor dimerization

The chemokine receptor genes of the CCR cluster on human chromosome 3p21 play important roles in humoral and cellular immune responses. Several of these receptors have been shown to influence human immunodeficiency virus infection and progression to AIDS, and their homologues may play a role in feline immunodeficiency virus infection. We report the isolation and sequencing of a 150-kb domestic cat BAC clone containing the feline CCR genes CCR1, CCR2, CCR3, and CCR5 to further analyze these four receptor genes within the family Felidae. Comparative and phylogenetic analyses reveal evidence for historic gene conversion between the adjacent CCR2 and CCR5 genes in the Felidae and in three independent mammalian orders (Primates, Cetartiodactyla, and Rodentia), resulting in higher than expected levels of sequence similarity between the two paralogous genes within each order. The gene conversion was restricted to the structural (transmembrane) domains of the CCR2 and CCR5 genes. We also discovered a recent gene conversion event between the third extracellular loop of CCR2 and CCR5 genes that was fixed in Asian lions and found at low frequency in African lions (Panthera leo), suggesting that this domain may have an important functional role. Our results suggest that ongoing parallel gene conversion between CCR2 and CCR5 promotes receptor heterodimerization in independent evolutionary lineages and offers an effective adaptive strategy for gene editing and coevolution among interactive immune response genes in mammals.     

Efficient Production of 2,5-Diketo-d-Gluconate via Heterologous Expression of 2-Ketogluconate Dehydrogenase in Gluconobacter japonicus

2,5-Diketo-d-gluconate (2,5DKG) is a compound that can be the intermediate for d-tartrate and also vitamin C production. Although Gluconobacter oxydans NBRC3293 produces 2,5DKG from d-glucose via d-gluconate and 2-keto-d-gluconate (2KG), with accumulation of the product in the culture medium, the efficiency of 2,5DKG production is unsatisfactory because there is a large amount of residual d-gluconate at the end of the biotransformation process. Oxidation of 2KG to 2,5DKG is catalyzed by a membrane-bound flavoprotein-cytochrome c complex: 2-keto-gluconate dehydrogenase (2KGDH). Here, we studied the kgdSLC genes encoding 2KGDH in G. oxydans NBRC3293 to improve 2,5DKG production by Gluconobacter spp. The kgdS, kgdL, and kgdC genes correspond to the small, large, and cytochrome subunits of 2KGDH, respectively. The kgdSLC genes were cloned into a broad-host-range vector carrying a DNA fragment of the putative promoter region of the membrane-bound alcohol dehydrogenase gene of G. oxydans for expression in Gluconobacter spp. According to our results, 2KGDH that was purified from the recombinant Gluconobacter cells showed characteristics nearly the same as those reported previously. We also expressed the kgdSLC genes in a mutant strain of Gluconobacter japonicus NBRC3271 (formerly Gluconobacter dioxyacetonicus IFO3271) engineered to produce 2KG efficiently from a mixture of d-glucose and d-gluconate. This mutant strain consumed almost all of the starting materials (d-glucose and d-gluconate) to produce 2,5DKG quantitatively as a seemingly unique metabolite. To our knowledge, this is the first report of a Gluconobacter strain that produces 2,5DKG efficiently and homogeneously.     

Geographic variation of color polymorphism in two sibling ladybird species,Harmonia yedoensis and H. axyridis (Coleoptera: Coccinellidae)

Geographical variation of elytra color pattern in two sibling ladybird species, Harmonia yedoensis and H. axyridis (Coleoptera: Coccinellidae), was examined. The two species are distributed sympatrically in central Japan; however, only H. yedoensis and H. axyridis occur in the Ryukyu Islands (southern Japan) and Hokkaido island (northern Japan), respectively. The frequency of elytra color patterns was significantly different between the two species in all sympatric locations and our results were inconsistent with the classical theory on Müllerian mimicry. The most dominant pattern of H. axyridis was the least dominant of H. yedoensis in all sympatric populations. Furthermore, the frequency of the non‐melanic form (red ground color with or without black spots) increased towards the south in H. yedoensis. This tendency was in contrast to the known geographical cline in H. axyridis in which the melanic form (black ground color with red spots) was gradually displaced with the non‐melanic form northwards in the Japanese archipelago. We discuss possible selective factors including predator avoidance, thermal adaptation and reproductive character displacement, all of which might contribute to the maintenance of the color polymorphism in the two Harmonia species.     

Design and synthesis of tricyclic tetrahydroquinolines as a new series of nonsteroidal selective androgen receptor modulators (SARMs)

Some tricyclic tetrahydroquinolines (THQs) were found to have the potential of a new series of nonsteroidal selective androgen receptor modulators (SARMs). Compound 5b was first designed and synthesized under our hypothesis based on a four-point pharmacophoric requirement of the 3-carbonyl, 18-methyl, 17-hydroxyl, and 13-quaternary carbon groups of dihydrotestosterone (DHT). It was revealed that this compound exhibits not only a strong androgen receptor (AR) agonistic activity (EC₅₀ = 9.2 nM) but also the highest selectivity in binding affinity to AR among the steroid hormone receptors. Furthermore, this compound showed a weak virilizing effect with retention of the desired anabolic effect as compared with DHT in vivo.     

Tetrahydroquinolines as a novel series of nonsteroidal selective androgen receptor modulators: structural requirements for better physicochemical and biological properties

A rationally designed tetrahydroquinoline (1) for nonsteroidal selective androgen receptor modulators was modified for the exploration of promising compounds by Grieco three-component condensation using various dienophiles. Based on the in vitro effects and physicochemical properties of the synthesized compounds, compound 4c was selected for further study. Compound 4c increased the femoral bone mineral density as much as DHT, but it reduced the uterus effect compared with DHT in ovariectomized rats. Thus, compound 4c has desirable osteoanabolic effects with weak undesirable effects on the uterus in a female osteoporosis model.     

6,7-Dihydro-5H-cyclopenta[d]pyrazolo[1,5-a]pyrimidines and their derivatives as novel corticotropin-releasing factor 1 receptor antagonists

To identify an orally active corticotropin-releasing factor 1 receptor antagonist, a series of 6,7-dihydro-5H-cyclopenta[d]pyrazolo[1,5-a]pyrimidines and their derivatives were designed, synthesized and evaluated. An in vitro study followed by in vivo and pharmacokinetic studies of these heterotricyclic compounds led us to the discovery of an orally active CRF1 receptor antagonist. The results of a structure-activity relationship study are presented.     

A transmembrane glycoprotein, gp38, is a novel marker for immature hepatic progenitor cells in fetal mouse livers

Previously, we clarified the surface antigen profiles of hepatic progenitor cells (HPCs) in fetal liver tissue as the CD49f(+)CD45(-)Thy1(-) cell fraction. However, these cells were a heterogeneous cell population containing various stages of differentiation. This study aimed to detect more immature HPCs, using a novel surface antigen, gp38. After the collagenase digestion of fetal livers harvested from E13.5 to E18.5 fetal mice, HPCs were obtained and divided into two subpopulations using flow cytometry: gp38-positive HPCs, and gp38-negative HPCs. Both types of HPCs were characterized by immunocytochemistry and RT-PCR. The proliferative activity was compared by BrdU incorporation and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTS) assay. Furthermore, the comprehensive gene expression was investigated by DNA microarray. Both types of HPCs expressed alpha-fetoprotein. However, the gp38-positive HPCs derived from E13.5 fetal livers did not express albumin or cytokeratin 19, while the gp38-negative HPCs did. DNA microarray revealed that some genes related to the Wnt signal pathway were up-regulated in the gp38-positive HPCs. Furthermore, Wnt3a had a proliferative effect on the gp38-positive HPCs. In conclusion, the gp38-positive HPCs derived from fetal liver tissue until E13.5 could therefore be candidates for hepatic stem cells in the fetal liver.