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Introduction of a methoxymethyloxy (MOMO) group at one of the four quinoline rings of TQEN (N,N,N′,N′-tetrakis-(2-quinolylmethyl)ethylenediamine) affords Cd2+-selective fluorescence enhancement.

Moderate Zn

EGTA (ethylene glycol bis(2-aminoethyl ether)-N,N,N′,N′-tetraacetic acid) and BAPTA (1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid) are well-known Ca2+ chelators that have four carboxylates, two nitrogen atoms and two ether oxygen atoms. In the present study, we prepared EGTQ (N,N,N′,N′-tetrakis(2-quinolylmethyl)-1,2-bis(2-aminoethoxy)ethane) and BAPTQ (N,N,N′,N′-tetrakis(2-quinolylmethyl)-1,2-bis(2-aminophenoxy)ethane) as quinoline alternatives of EGTA and BAPTA, respectively. In methanol–HEPES buffer solution (9 : 1, 50 mM HEPES, 0.1 M KCl, pH = 7.5), EGTQ exhibits fluorescence enhancement induced by Zn2+ and Cd2+ with poor selectivity, but BAPTQ did not exhibit a fluorescence response to either metal ion. Introduction of three methoxy substituents at the 5,6,7-positions of each quinoline moiety in BAPTQ specifically enhanced the fluorescence intensity of the Cd2+ complex, establishing the Cd2+-specific probe TriMeOBAPTQ (N,N,N′,N′-tetrakis(5,6,7-trimethoxy-2-quinolylmethyl)-1,2-bis(2-aminophenoxy)ethane). In contrast, TriMeOEGTQ (N,N,N′,N′-tetrakis(5,6,7-trimethoxy-2-quinolylmethyl)-1,2-bis(2-aminoethoxy)ethane) maintains a poor Cd2+/Zn2+ selectivity in its fluorescence response. Although the crystal structures of Cd2+/Zn2+ complexes with EGTQ and BAPTQ derivatives reveal the formation of multiple components including mononuclear and dinuclear complexes, the dinuclear Cd2+ and Zn2+ complexes with a linearly extended structure are regarded as possible fluorescent species in the solution. The conformational restriction of BAPTQ due to the orthophenylene moieties in the molecular skeleton is responsible for the formation of the weakly fluorescent, OH-bridged dizinc complex, which is critical to the strict Cd2+-specificity in the fluorescence response of TriMeOBAPTQ.

SummaryBackground Epidermolysis bullosa acquisita is a rare, potentially devastating autoimmune disease of the skin. IgG autoantibodies directed against type VII collagen (Col7), the major component of anchoring fibrils, induce skin fragility leading to cutaneous and mucocutaneous blister formation which is mostly of a scaring phenotype. Thus, powerful and reproducible diagnostic assays are critical to establish the diagnosis of EBA early to avoid irreversible sequelae. Objectives The present international, retrospective multicenter study includes a large cohort of EBA patients and evaluated the diagnostic power of four different diagnostic assays for the detection of anti-Col7 IgG autoantibodies. Methods 95 EBA sera and 200 control sera consisting of 100 bullous pemphigoid sera, 50 pemphigus vulgaris sera and 50 sera of healthy controls were tested for anti-Col7 IgG autoantibodies by indirect immunofluorescence (IIF), two commercial ELISA systems and Western blot (WB) analysis. EBA sera were from patients with positive DIF and IgG reactivity in at least one of the immune serological assays (IIF, ELISA, WB). Results A Col7-NC1/NC2 ELISA (MBL, Japan) showed the highest sensitivity (97.9%), followed by a Col7-NC1 ELISA (Euroimmun, Germany; 89.5%), WB with Col7-NC1 (85.3%), and IIF on saline split human skin (74.7%). The specificities of both ELISA systems were comparable (NC1: 98.7%, NC1/NC2: 99.3%). Furthermore, WB was more sensitive than IIF which was more specific. Conclusions The two commercially available ELISA systems allow for a highly sensitive and specific diagnosis of EBA. The sensitivity of the Col7-NC1/NC2 ELISA is significantly higher compared to the ELISA which is based on the Col7-NC1-domain only. This article is protected by copyright. All rights reserved.

A quinoline-based heptadentate ligand, N,N,N',N'-tetrakis(2-quinolylmethyl)-2,6-bis(aminomethyl)pyridine (TQLN), exhibits a Zn2+-specific fluorescence increase at 428 nm, which is assigned to excimer emission (IZn/I0 = 38, ICd/IZn = 24%, ϕZn = 0.069). In contrast, the isoquinoline counterpart 1-isoTQLN exhibits a Cd2+-specific fluorescence increase at 365 nm attributable to monomer emission (ICd/I0 = 83, IZn/ICd = 19%, ϕCd = 0.015).

EGTA (ethylene glycol bis(2-aminoethyl ether)-N,N,N',N'-tetraacetic acid) and BAPTA (1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid) are well-known Ca2+ chelators that have four carboxylates, two nitrogen atoms and two ether oxygen atoms. In the present study, we prepared EGTQ (N,N,N',N'-tetrakis(2-quinolylmethyl)-1,2-bis(2-aminoethoxy)ethane) and BAPTQ (N,N,N',N'-tetrakis(2-quinolylmethyl)-1,2-bis(2-aminophenoxy)ethane) as quinoline alternatives of EGTA and BAPTA, respectively. In methanol-HEPES buffer solution (9 : 1, 50 mM HEPES, 0.1 M KCl, pH = 7.5), EGTQ exhibits fluorescence enhancement induced by Zn2+ and Cd2+ with poor selectivity, but BAPTQ did not exhibit a fluorescence response to either metal ion. Introduction of three methoxy substituents at the 5,6,7-positions of each quinoline moiety in BAPTQ specifically enhanced the fluorescence intensity of the Cd2+ complex, establishing the Cd2+-specific probe TriMeOBAPTQ (N,N,N',N'-tetrakis(5,6,7-trimethoxy-2-quinolylmethyl)-1,2-bis(2-aminophenoxy)ethane). In contrast, TriMeOEGTQ (N,N,N',N'-tetrakis(5,6,7-trimethoxy-2-quinolylmethyl)-1,2-bis(2-aminoethoxy)ethane) maintains a poor Cd2+/Zn2+ selectivity in its fluorescence response. Although the crystal structures of Cd2+/Zn2+ complexes with EGTQ and BAPTQ derivatives reveal the formation of multiple components including mononuclear and dinuclear complexes, the dinuclear Cd2+ and Zn2+ complexes with a linearly extended structure are regarded as possible fluorescent species in the solution. The conformational restriction of BAPTQ due to the orthophenylene moieties in the molecular skeleton is responsible for the formation of the weakly fluorescent, OH-bridged dizinc complex, which is critical to the strict Cd2+-specificity in the fluorescence response of TriMeOBAPTQ.

In this study, we generated a new set of monoclonal antibodies (mAbs) to bovine and human type VII collagen (COL7) by immunizing mice with bovine cornea-derived basement membrane zone (BMZ) fraction. The four mAbs, tentatively named as COL7-like mAbs, showed speckled subepidermal staining in addition to linear BMZ staining of normal human skin and bovine cornea, a characteristic immunofluorescence feature of COL7, but showed no reactivity with COL7 by in vitro biochemical analyses. Taking advantage of the phenomenon that COL7-like mAbs did not react with mouse BMZ, we compared immunofluorescence reactivity between wild-type and COL7-rescued humanized mice and found that COL7-like mAbs reacted with BMZ of COL7-rescued humanized mice. In ELISAs, COL7-like mAbs reacted with intact triple-helical mammalian recombinant protein (RP) of COL7 but not with bacterial RP. Furthermore, COL7-like mAbs did not react with COL7 within either cultured DJM-1 cells or basal cells of skin of a bullous dermolysis of the newborn patient. These results confirmed that COL7-like mAbs reacted with human and bovine COL7. The epitopes for COL7-like mAbs were considered to be present only on mature COL7 after secretion from keratinocytes and deposition to BMZ and to be easily destroyed during immunoblotting procedure. Additional studies indicated association of the speckled subepidermal staining with both type IV collagen and elastin. These unique anti-COL7 mAbs should be useful in studies of both normal and diseased conditions, particularly dystrophic epidermolysis bullosa, which produces only immature COL7.

A quinoline-based heptadentate ligand, N,N,N',N'-tetrakis(2-quinolylmethyl)-2,6-bis(aminomethyl)pyridine (TQLN), exhibits a Zn

Plasma D-dimer (DD) is considered to be one of the most useful markers in the diagnosis and assessment of disseminated intravascular coagulation (DIC). The present study was performed to clarify the role of DD in a rat model of lipopolysaccharide (LPS)-induced DIC in which low-molecular-weight heparin (LMWH) and tranexamic acid (TA) were used. We investigated whether a relationship exists between plasma DD levels and severity of DIC. Experimental DIC was induced in rats by a sustained 4-hour infusion of 30 mg/kg LPS administered via the tail vein (LPS group). Rats received either LPS alone (LPS group) or LPS combined with 200 U/kg LMWH (LPS+LMWH group) or 50 mg/kg TA (LPS+TA group) from -30 minutes to 4 hours. Blood was drawn from each rat at 4, 8, and 12 hours. Plasma levels of thrombin-antithrombin complex (TAT) and creatinine were suppressed in the LPS+LMWH group, and less glomerular fibrin deposition was observed compared with the LPS group. On the other hand, an increased level of creatinine and increased glomerular fibrin deposition were observed in the LPS+TA group compared with the LPS group. LMWH demonstrated a protective effect against LPS-induced DIC, resulting in increased survival at 12 hours, whereas TA had the opposite effect. From these results, it appears that LMWH protects against LPS-induced DIC, but TA exacerbates LPS-induced DIC. It was interesting that plasma levels of DD were almost completely suppressed by concurrent administration of either TA or LMWH in this LPS-induced DIC model. This finding suggested that plasma levels of DD were suppressed by inhibition of coagulation (reduced deposition of fibrin) in the LPS+LMWH group and that DD levels were also suppressed by inhibition of fibrinolysis (reduced degradation of fibrin by plasmin) in the LPS+TA group. Thus care should be taken when evaluating the significance of plasma DD levels, because suppressed levels can occur with progressive fibrin deposition and worsening organ dysfunction or improvement in the course of DIC.

SummaryAlthough sepsis-induced release of nitric oxide (NO) is known to have an antithrombotic effect, it is unknown if NO exerts this same effect under physiological conditions. We have therefore attempted to determine whether or not NO protects against thrombus formation in normal Wistar rats injected with various amounts (0.8, 4.0, 20.0 and 100mg/kg/4hr) of L-NAME (N (omega)-nitro-l-arginine methyl ester), an NO synthase inhibitor, via the tail vein. Plasma levels of D-dimer fragments of fibrin were significantly increased in rats receiving L-NAME (0.21±0.04, 0.22±0.05, 0.26±0.07, 0.59±0.17µg/mL, means±SE; p

A 31-year-old man who underwent chemotherapy and bone marrow transplantation to treat acute myeloblastic leukemia was admitted to our department complaining of high fever and hypotension. His physical examination revealed warm shock state, eruptions resembling that seen in systemic lupus erythematosus on his face and cyanosis in his fingers. We diagnosed septic shock and idiopathic skin eruption on his face. Following treatment with blood transfusion, anticoagulant, antibiotics, respirator and continuous arteriovenous hemofiltration and dialysis, the patient's condition gradually improved. The eruptions on his face first observed at admission progressed with a worsening of his disseminated intravascular coagulation (DIC), and subsided with an improvement in his DIC. A biopsy of the eruption was taken and pathological findings of the eruption revealed multiple micro-fibrin depositions of the dermis. The skin necrosis in purpura fulminans often begins in the distal extremities. But our patient developed this uncommon skin eruption on his face. Patients with an idiopathic skin eruption resembling a butterfly rash in a septic patient should be considered to complicate DIC as in the present case.