Inta Kalnina

Spectroscopic Study of Benzanthrone 3-N-Derivatives as New Hydrophobic Fluorescent Probes for Biomolecules

We report the results on the spectroscopic properties of a new fluorescent lipophilic probes. Basic photophysical characteristics of the novel benzanthrone 3-amino-derivatives such as the absorption and fluorescence maxima, extinction coefficient, Stokes shift, fluorescence intensity were measured in benzene, chloroform and ethanol solutions. Novel benzanthrone 3-N-derivatives show bright fluorescence and are quite sensitive to the surrounding environment. The behaviour of the investigated benzanthrone derivatives was dependent on the polarity of the medium showing strong fluorescent solvatochromism arising from the donor–acceptor nature of the benzanthrone carbonyl group and electron-rich substituted amino group.

Fluorescent Probe ABM for Screening Gastrointestinal Patient’s Immune State

The fluorescent probe-aminoderivative of benzanthrone, ABM (developed at Riga Technical University, Riga, Latvia) was used to characterize the membranes of lymphocytes of cancer patients: 46 patients with gastrointestinal diseases, 13 patients having different primary localizations with massive metastases and intoxication. Patients were divided into three groups: (1) with decreased fluorescence intensity, (2) normal fluorescence intensity, (3) increased fluorescence intensity. The lymphocytes distribution among subsets differed between groups, in correspondence to the level of florescence intensity. Surgical treatment affected the main immunological parameters and elevated the functional activity of lymphocytes. In the advanced tumors group, fluorescence intensity correlates with the survival rate. Results suggest that determination of lymphocytes functional activity by ABM can aid evaluation of the immune status in cancer patients.

Correlation of altered blood albumin characteristics and lymphocyte populations to tumor stage in gastrointestinal cancer patients

The fluorescent probe ABM was used to characterize lymphocyte membranes and blood plasma albumin from cancer patients suffering from colorectal cancer or gastric cancers at Stages II-IV. The aim of these studies was to evaluate the potential utility of measures of ABM fluorescence intensity as a standard tool in the analyses of host immune status and for a clinical interpretation of alterations in albumin per se and lymphocyte functional activity in cancer patients. The fluorescence intensity of ABM in the blood plasma decreased from control values and showed specific differences in each of the differing patients groups; these changes corresponded to cancer stage. The significant decrease in ABM fluorescence in the plasma could be explained, in part, by a diminished binding capacity of the albumin of these patients. The lymphocyte distribution among the subsets of patients also differed. Interestingly, the ABM fluorescence in the cell suspension and blood plasma was also found to correlate with select immunological parameters (CD4(+):CD8(+) ratios, lymphocyte counts, etc.) in the patients. These results obtained here showed that there was a strong agreement between changes in ABM spectral characteristics and both clinical and pathological estimates of disease (i.e., gastrointestinal cancers) severity. Thus, the use of ABM spectroscopy appears to be another tool that might be of some used by clinicians to monitor the course of certain diseases, such as gastrointestinal cancers.

Fluorescence Investigation of Interactions Between Novel Benzanthrone Dyes and Lysozyme Amyloid Fibrils

A series of novel fluorescent benzanthrone dyes have been tested for their ability to identify and characterize fibrillar aggregates of lysozyme prepared by protein denaturation in concentrated ethanol solution (Feth) or acidic buffer (Fac). Quantitative parameters of the dye association with native and fibrillar protein have been derived from the results of fluorimetric titration. The binding characteristics proved to be different for Feth- and Fac-bound benzanthrones, highlighting the dye sensitivity to the distinctions in fibril morphology. By comparing the dye preference to fibrillar protein aggregates, AM2, A8 and A6 were selected as the most prospective amyloid tracers. Based on the analysis of red edge excitation shifts and fluorescence lifetimes of the amyloid-bound dyes it was assumed that surface grooves or dry “steric zipper” interface are potential fibril binding sites for the novel fluorophores.

Novel benzanthrone aminoderivatives for membrane studies.

The potential of novel benzanthrone aminoderivatives to trace the changes in physicochemical properties of lipid bilayer has been evaluated. Binding of the dyes to the lipid bilayers composed of zwitterionic phospholipid phosphatidylcholine (PC) and its mixtures with anionic phospholipid cardiolipin (CL) and cholesterol (Chol) was followed by significant quantum yield increase with small blue shift of emission maximum. Analysis of partition coefficients of the dyes under study showed that all aminobenzanthrones possess high lipid-associating ability. The dyes A8 and AM2 proved to be sensitive to the variations in membrane chemical composition responding to the changes in bilayer hydration induced by CL and Chol.

Structural Changes in Lymphocytes Membrane of Chernobyl Clean-up Workers from Latvia

ABM (3-aminobenzanthrrone derivative) developed at the Riga Technical University, Riga, Latvia) has been previously shown as a potential probe for determination of the immune state of patients with different pathologies .The fist study (using probe ABM) of peripheral blood mononuclear cells (PBMC) membranes of 97 Chernobyl clean-up workers from Latvia was conducted in 1997. Now we repeatedly examine the same (n = 54) individuals in dynamics. ABM spectral parameters in PBMC suspension, fluorescence anisotropy and blood plasma albumin characteristics were recorded. In 1997 screening showed 5 different patterns of fluorescence spectra, from which in 2007 we obtained only two. These patterns of spectra had never been previously seen in healthy individuals or patients with tuberculosis, multiple sclerosis, rheumatoid arthritis, etc., examined by us. Patterns of ABM fluorescence spectra are associated with membrane anisotropy and conformational changes of blood plasma albumin. We observed that in dynamics 1997–2007 the lipid compartment of the membrane became more fluid while the lipid-protein interface became more rigid. The use of probe ANS and albumin auto-fluorescence allowed show conformational alterations in Chernobyl clean-up workers blood plasma. It is necessary to note that all investigated parameters significantly differ in observed groups of patients. These findings reinforce our understanding that that the cell membrane is a significant biological target of radiation. The role of the membrane in the expression and course of cell damage after radiation exposure must be considered. So ten years dynamic of PBMC membrane characteristics by ABM (spectral shift and anisotropy indexes) in Chernobyl clean-up workers reveal progressive trend toward certain resemblance with those of chronic B-cell lymphoid leukemia.

Fluorescent Study of Human Blood Plasma Albumin Alterations Induced by Ionizing Radiation

The use of hydrophobic fluorescent probe ABM (benzanthrone derivative) and albumin autofluorescence allowed show conformational alterations in Chernobyl clean-up workers blood plasma. Results obtained in 1996–1997 suggest that acidic expansion of plasma albumin takes place. Latest data (2006–2008) result in splitting of albumin alterations onto two stages - acidic expansion and N-F transition. The N-F transition is accompanied by the blue shift of fluorescence spectra and dehydration of tryptophanyl region of albumin molecule. In 2007 obtained.patterns of ABM spectra had never been previously seen in examined healthy individuals or patients with tuberculosis, multiple sclerosis, rheumatoid arthritis, etc. Patterns of ABM fluorescence spectra are associated with conformational changes of blood plasma albumin. The use of probe ABM and albumin auto-fluorescence allowed show conformational alterations in albumin of Chernobyl clean-up workers blood plasma. It is necessary to note that all investigated parameters significantly differ in observed groups of patients. These findings reinforce our understanding that the blood plasma albumin is a significant biological target of radiation. It may be concluded that fluorescence characteristics are representative of radiation induced albumin alterations and its carrier function.

Altered plasma albumin characteristics and lymphocyte populations in gastrointestinal cancer patients: Detection using modified fluorescence responses

The fluorescent probe ABM (an amino derivative of benzanthrone) developed at Riga Technical University (Riga, Latvia) was used to characterize the blood plasma albumin of cancer patients (46 gastrointestinal cancer patients—30 with colorectal cancer in Stages II–IV and 16 with gastric cancer in Stage III) and of healthy controls. The fluorescence intensity of ABM in the blood plasma decreased from the control mean value and showed specific differences in the patients before (i.e., 24 hr pre-) and after (i.e., 10 day post-) they underwent a medically indicated surgical treatment, i.e., a gastric resection or gastroectomy for patients in the gastric cancer group or a colorectal resection for patients in the colorectum cancer group. The significant decrease in the ABM fluorescence in the blood plasma could be explained, in part, by a diminished binding capacity of the albumin of these patients. In fact, before surgery, there was a strong reduction in binding constant (Ka) value for the probe observed in the plasma samples from these patients as compared with the value obtained with the plasma of the healthy donors. The lymphocyte distribution among the subsets also differed between the groups. Surgical treatment affected several immunological parameters and appeared to elevate the functional status of lymphocytes. Interestingly, the ABM fluorescence in the blood plasma was also seen to correlate with select immunological parameters (CD4+:CD8+ ratios, levels of CD38+ cells, etc.) both before and after the patients’ operations. The results in the present study suggest that measures of ABM fluorescence intensity values for plasma albumin and/or especially of lymphocytes (as reflections of their functional activity) might be a useful tool in the evaluation of the immune status of gastrointestinal cancer patients.

Location of Novel Benzanthrone Dyes in Model Membranes as Revealed by Resonance Energy Transfer

Förster resonance energy transfer (FRET) between anthrylvinyl-labeled phosphatidylcholine (AV-PC) as a donor and newly synthesized benzanthrones (referred to here as A8, A6, AM12, AM15 and AM18) as acceptors has been examined to gain insight into molecular level details of the interactions between benzanthrone dyes and model lipid membranes composed of zwitterionic lipid phosphatidylcholine and its mixtures with anionic lipids cardiolipin (CL) and phosphatidylglycerol (PG). FRET data were quantitatively analyzed in terms of the model of energy transfer in two-dimensional systems taking into account the distance dependence of orientation factor. Evidence for A8 location in phospholipid headgroup region has been obtained. Inclusion of CL and PG into PC bilayer has been found to induce substantial relocation of A6, AM12, AM15 and AM18 from hydrophobic membrane core to lipid-water interface.

Antioxidant therapy and its stability on Chernobyl clean-up workers

This paper describes the effects of prolonged antioxidant therapy, its stability and association with spectral parameters of the fluorescent probe ABM in blood plasma and indices of albumin auto-fluorescence in Chernobyl clean-up workers from Latvia. Patients were divided into two groups. Group 1 received placebos of identical appearance to antioxidants. Group 2 received antioxidants (vitamin E + selenium + ibuprofen). Individuals were tested three times: before supplementation of antioxidants, after therapy and one year after supplementation interruption. Applied therapy improves oxidant/antioxidant status of individuals. Interruption of therapy after one year leads to significant decrease of cellular immunity. In the group receiving antioxidants it is not so expressed compared with data from the placebos group. Changes in blood plasma and its structural and functional properties detected by ABM strongly correlated with the oxidant/antioxidant (immune) status of individuals. This behaviour of ABM could be useful in reflecting alterations of cellular immunity. Questions related to more detailed and prolonged antioxidant therapy need closer investigation.