Reduced light exposure mitigates streptozotocin-induced vascular changes and gliosis in diabetic retina by an anti-inflammatory effect and increased retinal cholesterol turnover.

Diabetic retinopathy is not cured efficiently and changes of lifestyle measures may delay early retinal injury in diabetes. The aim of our study was to investigate the effects of reduced daily light exposure on retinal vascular changes in streptozotocin (STZ)-induced model of DM with emphasis on inflammation, Aqp4 expression, visual cycle and cholesterol metabolism-related gene expression in rat retina and RPE. Male Wistar rats were divided into the following groups: 1. control; 2. diabetic group (DM) treated with streptozotocin (100 mg/kg); 3. group exposed to light/dark cycle 6/18 h (6/18); 4. diabetic group exposed to light/dark cycle 6/18 h (DM+6/18). Retinal vascular abnormalities were estimated based on lectin staining, while the expression of genes involved in the visual cycle, cholesterol metabolism, and inflammation was determined by qRT-PCR. Reduced light exposure alleviated vasculopathy, gliosis and the expression of IL-1 and TNF-α in the retina with increased perivascular Aqp4 expression. The expression of genes involved in visual cycle and cholesterol metabolism was significantly up-regulated in RPE in DM+6/18 vs. DM group. In the retina only the expression of APOE was significantly higher in DM+6/18 vs. DM group. Reduced light exposure mitigates vascular changes and gliosis in DM via its anti-inflammatory effect, increased retinal cholesterol turnover and perivascular Aqp4 expression.

Investigation of the interaction between S-isoalkyl derivatives of the thiosalicylic acid and human serum albumin.

S-isoalkyl derivatives of thiosalicylic acid (isopropyl-(L1), isobutyl-(L2) and isoamyl-(L3)) were selected in order to investigate the binding interaction with the human serum albumin (HSA) using different spectroscopic methods and molecular docking simulation. Association constants and number of binding sites were used to analyze the quenching mechanism. The experimental results showed that the fluorescence quenching of HSA by L1, L2 and L3 occurs because of static quenching and that binding processes were spontaneous, with the leading forces in bonding by hydrogen bonding, hydrophobic interactions, and electrostatic interactions. Fluorescence spectroscopy, UV-Vis spectroscopy and synchronous fluorescence spectroscopy showed that ligands (L1, L2 and L3) can bind to HSA and that the binding of ligands induced some microenvironmental and conformational changes in HSA. The calculated distance between the donor and the acceptor according to fiFörster's theory confirms the energy transfer efficiency between the acceptor and HSA. Results of site marker competitive experiments showed that the tested compounds bind to HSA in domain IIA (Site I). Molecular dynamics and docking calculations demonstrated that L3 binds to the Sudlow site I of HSA with lower values of binding energies compared to L1 and L2, indicating the formation of the most stable ligand-HSA complex. Understanding the binding mechanisms of S-isoalkyl derivatives of the thiosalicylic acid to HSA may provide valuable data for the future studies of their biological activity and application as potential antitumor drugs.Communicated by Ramaswamy H. Sarma.

Complexes of copper(II) with tetradentate S,O-ligands: Synthesis, characterization, DNA/albumin interactions, molecular docking simulations and antitumor activity.

Four new complexes of copper(II) with S,O-tetradentate ligands, derivatives of thiosalicylic acid, encompassing an ethylene-, propylene-, butylene- and pentylene- bridge, were synthesized and characterized by microanalysis, molecular conductance and infrared (IR) spectra. The structures were assumed based on the previously mentioned analyses and confirmed with the results of electron paramagnetic resonance (EPR) spectra. The reactivity of complexes towards L-methionine (L-Met), L-cysteine (L-Cys) and guanosine-5'-monophosphate (5'-GMP) was also examined. Complex C1 ([Cu(S,O-ethylene-thiosalicylic acid)(H2O)2]) containing two inert methylene groups in the side chain of ligand shows the highest reactivity, while the least reactive is complex C4 ([Cu(S,O-pentylene-thiosalicylic acid)(H2O)2]) with five methylene groups. All complexes showed the highest reactivity towards L-Met and the lowest reactivity towards 5'-GMP. The interactions of complexes C1-C4 with calf thymus DNA (ct-DNA) were examined by ultraviolet-visible (UV-Vis) absorption and fluorescence spectral studies, revealing good DNA interaction abilities. All synthesized complexes C1-C4 show to interact with human serum albumin (HSA) with high values of binding constants. Complexes interaction with DNA/HSA was also confirmed using molecular docking simulations. All synthesized complexes reduce viability of human colon, breast and lung cancer cells, evaluated by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) colorimetric technique. The complex [Cu(S,O-pentylene- thiosalicylic acid)(H2O)2] showed the highest binding affinity constants to DNA/HSA and highest cytotoxicity, thus presenting a good candidate for further pharmacological research in the field of colon, breast and lung cancer therapy.

Bioassays for anticancer activities.

The MTT/MTS in vitro cell proliferation assay is one of the most widely used assays for evaluating preliminary anticancer activity of both synthetic derivatives and natural products and natural product extracts. The highly reliable, colorimetric based assay is readily performed on a wide range of cell lines. This assay gives an indication of whole cell cytotoxicity; however, to determine the exact molecular target further assays need to be performed. Of these, kinase inhibition assays are also one of the most widespread enzyme inhibition screening assays performed. Kinases are enzymes that play a key role in a number of physiological processes and their inhibitors have been found to exhibit anticancer activity against various human cancer cell lines. Herein, we describe the methods for performing both in vitro MTT/MTS cytotoxicity and kinase enzyme inhibition assays. These are two of the most useful anticancer screening techniques available that are relatively economical and can be easily and routinely performed in the laboratory to characterize anticancer activity. Both assays are highly versatile and can be modified to test against targeted disease processes by using specific kinase enzymes or cell lines.

c-AMP dependent protein kinase A inhibitory activity of six algal extracts from southeastern Australia and their fatty acid composition.

c-AMP dependent protein kinase (protein kinase A, PKA) is an important enzyme involved in the regulation of an increasing number of physiological processes including immune function, cardiovascular disease, memory disorders and cancer. The objective of this study was to evaluate the PKA inhibitory activity of a range of algal extracts, along with their fatty acid composition. Six algal species were investigated including two Chlorophyta (Codium dimorphum and Ulva lactuca), two Phaeophyta (Phyllospora comosa and Sargassum sp.) and two Rhodophyta (Prionitis linearis and Corallina vancouveriensis), with the order of PKA inhibitory activity of their extracts identified as follows: brown seaweeds > red seaweeds > green seaweeds with the brown alga Sargassum sp. exhibiting the highest PKA inhibitory activity (84% at 100 microg/mL). GC/MS analysis identified a total of 18 fatty acids in the six algal extracts accounting for 72-87% of each extract, with hexadecanoic acid and 9,12-octadecadienoic acid as the dominant components. The most active extract (Sargassum sp.) also contained the highest percentage of the saturated C14:0 fatty acid (12.8% of the total extract), which is a known to inhibit PKA. These results provide the first description of the PKA inhibitory activity of marine algae along with the first description of the fatty acid composition of these six algal species from South Eastern Australian waters. Importantly, this study reveals that abundant and readily available marine algae are a new and relatively unexplored source of PKA inhibitory compounds.

Kinase inhibitors from marine sponges.

Protein kinases play a critical role in cell regulation and their deregulation is a contributing factor in an increasing list of diseases including cancer. Marine sponges have yielded over 70 novel compounds to date that exhibit significant inhibitory activity towards a range of protein kinases. These compounds, which belong to diverse structural classes, are reviewed herein, and ordered based upon the kinase that they inhibit. Relevant synthetic studies on the marine natural product kinase inhibitors have also been included.

Kinase inhibitory, haemolytic and cytotoxic activity of three deep-water sponges from North Western Australia and their fatty acid composition.

The c-AMP dependent protein kinase (PKA) inhibition, haemolytic activity, and cytoxicity of 21 extracts obtained from North Western Australian sponges collected from depths of 84-135 m were investigated. Hexane extracts from Ircinia/Sarcotragus sp. and Geodia sp. displayed PKA inhibitory activities of 100 and 97% respectively (at 100 microg/mL), while aq. methanol extracts from Haliclona sp. exhibited potent haemolytic activity (75%) and hexane extracts from Geodia sp. were highly toxic (88%) to the brine shrimp Artemia franciscana. As the non-polar extracts gave the greatest PKA inhibition, these were further analysed by GC-MS and 29 fatty acids were identified in the highest proportions in Ircinia/Sarcotragus sp. > Haliclona sp. > Geodia sp. In contrast to shallow-water sponges that are dominated by polyunsaturated fatty acids with a high percentage of long chain fatty acids, LCFAs (C24-C30), the deep-sea sponges investigated herein were all found to be rich in saturated fatty acids, in particular C14-C20 fatty acids, including odd and branched chain fatty acids, with only low levels (0-10%) of LCFAs. Screening of the PKA inhibitory activity of a series of commercially available fatty acids identified C14-C18 fatty acids as possessing significant PKA inhibitory activity that may contribute to the activity observed in the sponges studied.