Protochlorophyllide forms and energy transfer in dark-grown wheat leaves. Studies by conventional and laser excited fluorescence spectroscopy between 10 K–100 K

The fluorescence properties and role in energy transfer of protochlorophyllide (Pchlide) forms were studied in dark-grown wheat leaves by conventional and laser excited high resolution methods in the 10 K–100 K temperature range. The three major spectral bands, with emission maxima at 633, 657 (of highest intensity) and 670 nm as Bands I, II, and III were analyzed and interpreted as the contributions of six different structural forms. Band I is the envelope of three (0,0) emission bands with maxima at 628, 632 and 642 nm. Laser excitation studies in the range of Band II at 10 K reveal the presence of a spectrally close donor band besides the acceptor, Band II. The intensity in Band III originates mostly from being the vibronic satellite of Band II, but contains also a small (0,0) band with absorption maximum at 674 nm. Excitation spectra show that besides the Pchlides with absorption around 650 nm within Band II, another significant population of Band I with absorption around 640 nm is also coupled by energy transfer to the acceptor of Band II. The spectral difference between the two donor forms indicate different dipolar environments. Upon increasing the temperature, the intensity of Band II and its satellite, Band III decrease, while Band I remains unaffected. Band II shows also a broadening towards the blue side at higher temperatures. Both the quenching of fluorescence and the spectral change was explained by a thermally activated formation of a non-fluorescent intermediate state in the excited state of Pchlide acceptors.     

Development of piperazine-based hydroxamic acid inhibitors against falcilysin,an essential malarial protease

The human parasite Plasmodium falciparum kills an estimated 445,000 people a year, with the most fatalities occurring in African children. Previous studies identified falcilysin (FLN) as a malarial metalloprotease essential for parasite development in the human host. Despite its essentiality, the biological roles of this protease are not well understood. Here we describe the optimization of a piperazine-based hydroxamic acid scaffold to develop the first reported inhibitors of FLN. Inhibitors were tested against cultured parasites, and parasiticidal activity correlated with potency against FLN. This suggests these compounds kill P. falciparum by blocking FLN, and that FLN is a druggable target. These compounds represent an important step towards validating FLN as a therapeutic target and towards the development of chemical tools to investigate the function of this protease.     

Morphological and proteomic analysis of early stage of osteoblast differentiation in osteoblastic progenitor cells

Bone remodeling relies on a dynamic balance between bone formation and resorption, mediated by osteoblasts and osteoclasts, respectively. Under certain stimuli, osteoprogenitor cells may differentiate into premature osteoblasts and further into mature osteoblasts. This process is marked by increased alkaline phosphatase (ALP) activity and mineralized nodule formation. In this study, we induced osteoblast differentiation in mouse osteoprogenitor MC3T3-E1 cells and divided the process into three stages. In the first stage (day 3), the MC3T3-E1 cell under osteoblast differentiation did not express ALP or deposit a mineralized nodule. In the second stage, the MC3T3-E1 cell expressed ALP but did not form a mineralized nodule. In the third stage, the MC3T3-E1 cell had ALP activity and formed mineralized nodules. In the present study, we focused on morphological and proteomic changes of MC3T3-E1 cells in the early stage of osteoblast differentiation — a period when premature osteoblasts transform into mature osteoblasts. We found that mean cell area and mean stress fiber density were increased in this stage due to enhanced cell spreading and decreased cell proliferation. We further analyzed the proteins in the signaling pathway of regulation of the cytoskeleton using a proteomic approach and found upregulation of IQGAP1, gelsolin, moesin, radixin, and Cfl1. After analyzing the focal adhesion signaling pathway, we found the upregulation of FLNA, LAMA1, LAMA5, COL1A1, COL3A1, COL4A6, and COL5A2 as well as the downregulation of COL4A1, COL4A2, and COL4A4. In conclusion, the signaling pathway of regulation of the cytoskeleton and focal adhesion play critical roles in regulating cell spreading and actin skeleton formation in the early stage of osteoblast differentiation.