Spectrophotometric determination of urinary protein with o-sulfophenylfluorone-metal complex.

A simple and sensitive spectrophotometric method for the determination of human serum albumin (HSA) was established based on the ternary complex-formation reaction of HSA with o-sulfophenylfluorone (SPF) as a xanthene dye and metal ion (niobium(V) and bismuth(III)) in the presence of a dispersion agent. This new method enabled the determination of HSA in the range of 1 - 15 microg/ml HSA by measuring the difference of the absorbance at 530 nm between HSA-SPF-metal ion and SPF-metal ion solutions. In the determination of HSA, this method is about 2-times more sensitive than the Pyrogallol Red-molybdenum(VI) method (PR method), which accounts for more than 80% of the quantification methods for urinary protein assays in Japan. There was no significant difference between the results obtained by the present method and the PR method for human urine samples. The binding process between the SPF-metal complex and HSA was studied by determining the binding parameters and the thermodynamic parameters.     

High-performance liquid chromatographic determination and metabolic study of sennoside a in daiokanzoto by mouse intestinal bacteria

Daiokanzoto (DKT, combination of rhubarb and glycyrrhiza), a Kampo medicine, is clinically effective for constipation. Sennoside A is well known to induce diarrhea. Sennoside A is a prodrug that is transformed into an active metabolite, rheinanthrone, by intestinal bacteria. In this study, we investigated the effects of glycyrrhiza on the activity of sennoside A metabolism in intestinal bacteria using mouse feces. A high-performance liquid chromatography (HPLC) method for the determination of sennoside A in incubation mixture of DKT with mouse feces was established. The retention time of sennoside A was 9.26±0.02 min with a TSKgel ODS-80TsQA column by linear gradient elution using a mobile phase containing aqueous phosphoric acid and acetonitrile and detection at 265 nm. We found that the activity of sennoside A metabolism in intestinal bacteria was significantly accelerated when glycyrrhiza, liquiritin or liquiritin apioside coexisted with sennoside A, whereas that of glycyrrhizin was not altered. This method is applicable for determination of the activity of sennoside A metabolism by anaerobic incubation of DKT with mouse feces.     

Photoinduced electron-transfer reaction of α-bromomethyl-substituted benzocyclic β-keto esters with amines: selective reaction pathways depending on the nature of the amine radical cations

Photoinduced electron-transfer reaction of α-bromomethyl-substituted benzocyclic β-keto esters with tertiary amines was investigated. Debrominated β-keto esters and ring-expanded γ-keto esters were obtained as major products. On the basis of mechanistic experiments it was concluded that these products are formed via a reaction sequence of selective carbon–bromine bond cleavage and subsequent competitive hydrogen abstraction and Dowd–Beckwith ring-expansion of the resulting primary alkyl radicals. The characteristic product distribution observed for the type of amine used is rationalized on the basis of selective reaction pathways of generated radical intermediates that depend on the nature of the amine radical cations.     

Novel Copolymers of Vinyl Acetate and Some Ring‐Substituted 2‐Phenyl‐1,1‐dicyanoethylenes

Electrophilic trisubstituted ethylene monomers, some ring‐substituted 2‐phenyl‐1,1‐dicyanoethylenes, RC₆H₄CH?C(CN)₂ (where R is 3‐Br, 4‐CH₃O; 5‐Br, 2‐CH₃O; 4‐Cl, 3‐NO₂; 5‐Cl, 2‐NO₂; 2‐CN, 3‐CN, 4‐CN, and 4‐(CH₃)₂N), were synthesized by piperidine catalyzed Knoevenagel condensation of ring‐substituted benzaldehydes and malononitrile, and characterized by CHN elemental analysis, IR, ¹H‐ and ¹³C‐NMR. Novel copolymers of the ethylenes and vinyl acetate were prepared at equimolar monomer feed composition by solution copolymerization in the presence of a radical initiator (ABCN) at 70°C. The composition of the copolymers was calculated from nitrogen analysis, and the structures were analyzed by IR, ¹H and ¹³C‐NMR, GPC, DSC, and TGA. High T _g of the copolymers, in comparison with that of polyvinyl acetate, indicates a substantial decrease in chain mobility of the copolymer due to the high dipolar character of the trisubstituted ethylene monomer unit. The gravimetric analysis indicated that the copolymers decompose in the 190–800°C range.     

Tetraspanin CD9 modulates ADAM17-mediated shedding of LR11 in leukocytes

LR11, also known as SorLA or SORL1, is a type-I membrane protein from which a large extracellular part, soluble LR11 (sLR11), is released by proteolytic shedding on cleavage with a disintegrin and metalloproteinase 17 (ADAM17). A shedding mechanism is presumed to have a key role in the functions of LR11, but the evidence for this has not yet been demonstrated. Tetraspanin CD9 has been recently shown to regulate the ADAM17-mediated shedding of tumor necrosis factor-α and intercellular adhesion molecule-1 on the cell surface. Here, we investigated the role of CD9 on the shedding of LR11 in leukocytes. LR11 was not expressed in THP-1 monocytes, but it was expressed and released in phorbol 12-myristate 13-acetate (PMA)-induced THP-1 macrophages (PMA/THP-1). Confocal microscopy showed colocalization of LR11 and CD9 proteins on the cell surface of PMA/THP-1. Ectopic neo-expression of CD9 in CCRF-SB cells, which are LR11-positive and CD9-negative, reduced the amount of sLR11 released from the cells. In contrast, incubation of LR11-transfected THP-1 cells with neutralizing anti-CD9 monoclonal antibodies increased the amount of sLR11 released from the cells. Likewise, the PMA-stimulated release of sLR11 increased in THP-1 cells transfected with CD9-targeted shRNAs, which was negated by treatment with the metalloproteinase inhibitor GM6001. These results suggest that the tetraspanin CD9 modulates the ADAM17-mediated shedding of LR11 in various leukemia cell lines and that the association between LR11 and CD9 on the cell surface has an important role in the ADAM17-mediated shedding mechanism.     

Elimination of ingredients effect to improve the detection of anti HIV-1 p24 antibody in human serum using SPR apparatus.

Highly sensitive detection of proteins in serum becomes difficult in some cases during surface plasmon resonance (SPR) measurements, because some ingredients in the serum hugely enhance non-specific reactions on the sensing chip of SPR. It is well recognized that the antibody against core protein p24 of HIV in serum is one of the most important proteins in the accurate diagnosis of infection with HIV. In this study, we could attain the accurate detection of anti p24 antibody in human serum by eliminating the serious effects of the ingredients in serum on the measurement of SPR by employing these procedures: 1) blocking the gold surface of the sensing chip with human serum and 2) heating the serum sample at 56 degrees C for 30 min. Without these treatments, the signal of SPR was considerably suppressed on the measurements of the anti p24 antibody which contained human serum, making the accurate detection difficult. However, with introducing the above two treatments, the sensing of anti p24 antibody in human serum was improved, while a small non-specific reaction was still observed. By removing the non-specific reaction caused by the ingredients in the serum, we could accurately measure the antibody for p24 in human serum sample over the range from 1 to 20 micrograms/ml.     

Catalytic phenylborylation reaction by iridium(0) nanoparticles produced from hydridoiridium carborane

Well-dispersed iridium(0) nanoparticles stabilized with the ionic liquid, trihexyltetradecylphosphonium methylsulfonate, [THTdP][MS], have been successfully prepared by reduction of the precursor hydridoiridium carborane, (Ph 3P) 2Ir(H)(7,8- nido-C 2B 9H 11). The iridium nanoparticles were found to be active catalysts for arylborylation, forming boric acids. The activity of the catalyst has been investigated as a function of the activating base, and reaction conditions. The highest yield of 91% was achieved in a microwave reactor using the base, tetra-2-pyridinylpyrazine, in the presence of [THTdP][MS]. The catalytic system could be recycled at least six times with less than a 0.5% loss of activity.     

Unusual condensation of 3-Thia[5](1,1')ferrocenophane-1,5-dione with glyoxal. Formation of [1.1](2,3)thiopheno(1,1')ferrocenophane-1,7-dione

When 3-thia [5](1,1')ferrocenophane-1,5-dione 2 was condensed with glyoxal in the presence of sodium ethoxide, a thiophene-2,3-diyl derivative 3 was obtained as a sole product, instead of the expected 2,5-substituted compound 1.