Competent dendritic cells derived from CD34+ progenitors express CMRF-44 antigen early in the differentiation pathway

Differentiation of CD34(+) haematopoietic stem cells into functional dendritic cells (DC) was investigated using the mAb CMRF-44 and other mAb against DC-associated markers. GM-CSF mobilized peripheral blood stem cells were obtained from healthy donors by leukapheresis. CD34(+) cells were purified using CD34(+)-positive selection,and subsequent immunomagnetic depletion of CD14 and CD2 cells. CD34(+) cells were cultured in medium supplemented with one or more of GM-CSF,TNF-alpha, IL-4 or IL-6. CMRF-44 Ag expression was monitored by flow cytometry, and DC function by allogeneic MLR and tetanus toxoid(TT) presentation assays. CD34(+) cells quickly acquired the CMRF-44 Ag when cultured in the presence of TNF-alpha.By day 3, more than 50% of the cells were double-positive for CD34 and CMRF-44. CD34 expression was gradually lost, so that by day 9, the majority of the cells were CD34(-)/CMRF-44(+).GM-CSF and TNF-alpha also induced CD40 expression, and up-regulation of CD54 and MHC class II on CD34(+) cells; their expression was correlated to the CMRF-44 Ag. Day 3 CD34(+)/CMRF-44(+) cells,but not CD34(+)/CMRF-44(-) cells, become potent APC when cultured further with GM-CSF plus TNF-alpha. These CMRF-44(+) cells were potent inducers of Th1-type immune response in the primary allogeneic MLR and present TT to autologous CD4(+) T cells. TNF-alpha alone is sufficient to induce CMRF-44 expression on CD34(+) cells, but in combination with GM-CSF expands the CMRF-44(+) population. CMRF-44 expression correlates with DC function and may be a useful early marker for commitment of CD34(+) cells to the DC differentiation pathway.     

Brain-derived Neurotrophic Factor (BDNF) Induces Sustained Intracellular Ca2+ Elevation through the Up-regulation of Surface Transient Receptor Potential 3 (TRPC3) Channels in Rodent Microglia

Microglia are immune cells that release factors, including proinflammatory cytokines, nitric oxide (NO), and neurotrophins, following activation after disturbance in the brain. Elevation of intracellular Ca²⁺ concentration ([Ca²⁺]_i) is important for microglial functions such as the release of cytokines and NO from activated microglia. There is increasing evidence suggesting that pathophysiology of neuropsychiatric disorders is related to the inflammatory responses mediated by microglia. Brain-derived neurotrophic factor (BDNF) is a neurotrophin well known for its roles in the activation of microglia as well as in pathophysiology and/or treatment of neuropsychiatric disorders. In this study, we sought to examine the underlying mechanism of BDNF-induced sustained increase in [Ca²⁺]_i in rodent microglial cells. We observed that canonical transient receptor potential 3 (TRPC3) channels contribute to the maintenance of BDNF-induced sustained intracellular Ca²⁺ elevation. Immunocytochemical technique and flow cytometry also revealed that BDNF rapidly up-regulated the surface expression of TRPC3 channels in rodent microglial cells. In addition, pretreatment with BDNF suppressed the production of NO induced by tumor necrosis factor α (TNFα), which was prevented by co-adiministration of a selective TRPC3 inhibitor. These suggest that BDNF induces sustained intracellular Ca²⁺ elevation through the up-regulation of surface TRPC3 channels and TRPC3 channels could be important for the BDNF-induced suppression of the NO production in activated microglia. We show that TRPC3 channels could also play important roles in microglial functions, which might be important for the regulation of inflammatory responses and may also be involved in the pathophysiology and/or the treatment of neuropsychiatric disorders.     

Quantitation of serum retinol-binding protein by an enzyme-linked immunosorbent assay

An enzyme-linked immunosorbent technique for human serum retinol-binding protein (RBP) was developed. The assay detects RBP via a double-antibody (rabbit anti-human RBP) sandwich technique. The antibody is immobilized by passive adsorption to a polystyrene tube; the assay is then carried out by successive additions containing known and unknown amounts of RBP (antigen), alkaline phosphatase linked to the same antibody, and p-nitrophenyl phosphate (substrate). Colorimetric analysis of the hydrolysis of the substrate by the enzyme (indirectly) attached to the antigen is used for RBP quantitation. The intra- and interassay coefficients of variation ranged between 4 and 7 and 9 and 12%, respectively. The assay can be performed in less than 7 h and has a sensitivity in the nanogram range (3–48 ng/ml). RBP content was analyzed in serum and urine samples of 20 healthy donors and 17 patients with renal failure and in 20 serum specimens of patients with liver cirrhosis. Renal patients had higher serum (mean 150, range 50–398 μg/ml) and urine RBP levels (mean 14, range 1–80 μg/ml) than normal donors (mean serum 43, range 30–60 μg/ml; mean urine RBP 0.06, range 0.04 – 0.13 μg/ml). Liver disease patients had lower than normal serum RBP values (mean 22, range 10–43 μg/ml).     

Amyloid formation in denatured single-mutant lysozymes where residual structures are modulated

Reduced hen lysozyme has a residual structure involving long-range interaction. It has been demonstrated that a single mutation (A9G, W62G, W111G, or W123G) in the residual structure differently modulates the long-range interactions of reduced lysozyme. To examine whether such variations in the residual structure affect amyloid formation, reduced and alkylated mutant lysozymes were incubated under the amyloid-fibrillation condition. From the analyses of CD spectra and thioflavine T fluorescences, it was suggested that variation in residual structure led to different amyloid formation. Interestingly, the extent of amyloid formation did not always correlate with the extent to which the residual structure was maintained, resulting in the involvement of a hydrophobic cluster normally contained in W111 in the reduced lysozyme.     

Residual Structures in the Acid-Unfolded States of Vλ6 Proteins Affect Amyloid Fibrillation

Many proteins form amyloid-like fibrils in vitro under partially or highly unfolding conditions. Recently, we showed that the residual structure in highly unfolded state is closely related to amyloid fibril formation in hen lysozyme. Thus, to better understand the role of the residual structure on amyloid fibril formation, we focused on AL amyloidosis, which results from the extracellular deposition of monoclonal immunoglobulin light-chain variable domains (V_Ls) as insoluble fibrils. We examined the relationship between the residual structure and amyloid fibril formation on three λ6 recombinant V_L (rVλ6) proteins, wild type, Jto, and Wil. Although rVλ6 proteins are highly unfolded in pH 2, ¹⁵N NMR transverse relaxation experiments revealed nonrandom structures in regions, which include some hydrophobic residues and a single disulfide bond, indicating the existence of residual structure in rVλ6 proteins. However, the residual structure of Wil was markedly disrupted compared with those of the other proteins, despite there being no significant differences in amino acid sequences. Fibrillation experiments revealed that Wil had a longer lag time for fibril formation than the others. When the single disulfide bond was reduced and alkylated, the residual structure was largely disrupted and fibril formation was delayed in all three rVλ6 proteins. It was suggested that the residual structure in highly unfolded state has a crucial role in amyloid fibril formation in many proteins, even pathogenic ones.     

Inhibitory effects of calcium ionophore pretreatment of porcine oocytes on polyspermic fertilization

The present study examined the inhibitory effects of various pretreatment concentrations (0-100 microM) of the calcium ionophore A23187 on polyspermic fertilization and then examined the effect of the maturation period and the time between calcium ionophore treatment and fertilization on the inhibitory effect of calcium ionophore on polyspermic fertilization. In experiment 1, a high concentration of calcium ionophore (100 microM) increased the rate of activated oocytes, but the rate of fertilization declined. On the other hand, when oocytes were treated with a low concentration of calcium ionophore (10 microM), monospermic fertilization was significantly increased (10 microM; 31.3%) (p < 0.05). In experiment 2, oocytes were cultured for various times (0, 0.5, 3, 6 h) after calcium ionophore treatment (10 microM) before fertilization. The highest rate of monospermic fertilization was detected in the oocytes cultured for 6 h after calcium ionophore treatment before fertilization. In experiments 3 and 4, we examined the effect of the maturation period (40 h or 44 h) on the rate of fertilization and blastulation of oocytes pretreated with calcium ionophore. The treatment of oocytes with calcium ionophore significantly decreased the rate of polyspermic fertilization regardless of the maturation period (44 h: with calcium ionophore 26.25% vs without 78.8%; 40 h: with calcium ionophore 37.5% vs without 77.5%); however, calcium ionophore treatment increased the rates of monospermic fertilization and blastulation of the oocytes matured for 44 h, but not those matured for 40 h. In conclusion, activation with a low concentration of calcium ionophore (10 microM) and a further 6 h of culture before fertilization improved the rate of monospermic fertilization and blastulation.     

Mechanism for retardation of amyloid fibril formation by sugars in Vλ6 protein

Sugars, which function as osmolytes within cells, retard the amyloid fibril formation of the amyloidosis peptides and proteins. To examine the mechanism of this retardation in detail, we analyzed the effect of sugars (trehalose, sucrose, and glucose) on the polypeptide chains in 3Hmut Wil, which is formed by the mutation of three His residues in Wil mutant as a cause of amyloid light‐chain (AL) amyloidosis, at pH 2, a pH condition under which 3Hmut Wil was almost denatured. Sugars caused the folding of 3Hmut Wil so that its polypeptide chains adopted a native‐like rather than a denatured conformation, as suggested by tryptophan fluorescence, CD spectroscopy, and heteronuclear NMR. Furthermore, these sugars promoted the folding to a native‐like conformation according to the effect of preferential hydration rather than direct interaction. However, the type of sugar had no effect on the elongation of amyloid fibrils. Therefore, it was concluded that sugar affected the thermodynamic stability of 3Hmut Wil but not the elongation of amyloid fibrils.     

Rapid detection of fluoroquinolone resistance by isothermal chimeric primer-initiated amplification of nucleic acids from clinical isolates of Neisseria gonorrhoeae

To ensure a complete response to fluoroquinolone therapy against Neisseria gonorrhoeae infections, rapid susceptibility determinations are required. We assessed a new approach, an isothermal chimeric primer-initiated amplification of nucleic acids (ICAN)/hybrid-chromatography method to detect rapidly fluoroquinolone resistance in N. gonorrhoeae. Comparison of the amplification results with fluoroquinolone minimum inhibitory concentrations (MICs), which were determined by an agar dilution method, showed that the new method accurately determined fluoroquinolone resistance in all ciprofloxacin- and/or gatifloxacin-resistant isolates, but agreed with results based on MICs in only 6 of 8 (75.0%) ciprofloxacin-susceptible and 7 of 12 (58.3%) gatifloxacin-susceptible isolates. Our results suggest that this method can rapidly and reliably detect point mutations in the gyrA gene as well as fluoroquinolone resistance in resistant isolates of N. gonorrhoeae.