Seminaphthofluorescein-Based Fluorescent Probes for Imaging Nitric Oxide in Live Cells

Fluorescent turn-on probes for nitric oxide based on seminaphthofluorescein scaffolds were prepared and spectroscopically characterized. The Cu(II) complexes of these fluorescent probes react with NO under anaerobic conditions to yield a 20–45-fold increase in integrated emission. The seminaphthofluorescein-based probes emit at longer wavelengths than the parent FL1 and FL2 fluorescein-based generations of NO probes, maintaining emission maxima between 550 and 625 nm. The emission profiles depend on the excitation wavelength; maximum fluorescence turn-on is achieved at excitations between 535 and 575 nm. The probes are highly selective for NO over other biologically relevant reactive nitrogen and oxygen species including NO3–, NO2–, HNO, ONOO–, NO2, OCl–, and H2O2. The seminaphthofluorescein-based probes can be used to visualize endogenously produced NO in live cells, as demonstrated using Raw 264.7 macrophages.National Science Foundation (U.S.) (CHE-0611944)National Institutes of Health (U.S.) (K99GM092970

Pathogenic Role of NF-κB Activation in Tubulointerstitial Inflammatory Lesions in Human Lupus Nephritis

In vitro and in vivo experimental studies suggest that the transcription factor NF-κB plays a role in tubulointerstitial injury. We investigated possible cellular and molecular mechanisms involving NF-κB activation in the progression of tubulointerstitial lesions in human lupus nephritis (LN). Paraffin-embedded renal biopsies from 50 patients with LN and six control patients with minimal change disease (MCD) were examined by Southwestern histochemistry for in situ detection of active NF-κB and AP-1. Immunohistochemistry was performed to examine the expression of NF-κB, AP-1, and NF-κB regulatory proteins (IκB-α, p-IκB-α, and IKK-α proteins), as well as NF-κB and AP-1 downstream target proinflammatory molecules (ICAM-1, TNF-α, IL-1β, IL-6, and GM-CSF) and NF-κB upstream signaling molecules (CD40 and CD40L). We observed extensive upregulation of activated NF-κB in renal tubular cells and interstitial cells, in parallel with overactivation of transcription factor AP-1 in LN, as compared with normal controls and MCD. Tubular expression of activated NF-κB correlated well with the degree of tubulointerstitial histopathological indices and/or renal function. Tubulointerstitial IKK-α expression was specifically upregulated in LN. IκB-α and p-IκB-α were detected only in interstitial cells in LN. Tubulointerstitial expression levels of NF-κB and AP-1 downstream inflammatory molecules and NF-κB upstream signaling molecules CD40 and CD40L were markedly enhanced in LN as compared with MCD or normal controls and were associated with tubulointerstitial histopathological indices and/or renal function. The results suggest that altered IKK-α expression and NF-κB activation along with AP-1 overexpression may play a pathogenic role in tubulointerstitial injury in human LN mediated through a network of downstream proinflammatory molecules. (J Histochem Cytochem 56:517–529, 2008